Independent helper virus packaging cell line for propagating viral vectors

ABSTRACT

Provided are novel vectors and viral vectors capable of expressing exogenous gene or exogenous nucleic acid sequences in a target cell of interest, such as T cells, bone marrow cells, epithelial cells, liver cells and the like. The nucleic acid components of the vectors may include one or more native promoter/enhancer regions having modified sequence segments, one or more non-native promoter/enhancer or non-native promoter&#39;s gene or gene segment, and a native viral vector terminator or processing signal or segment thereof. The viral vectors comprise a virus or viral portion having on the surfaces or envelopes adsorption components, one for a packaging cell line and the other for delivery to a target cell. Packaging cell lines for propagating the vectors and viral vectors are also provided, as are novel processes for propagating any of the disclosed vectors or viral vectors.

CROSS-REFERENCE TO OTHER RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.08/822,963, filed on Mar. 21, 1997 now abandoned.

FIELD OF THE INVENTION

This invention relates to the field of recombinant nucleic acidtechnology, and more particularly, to the production of gene expressionsystems involving novel vectors and viral vectors as well as uniquepackaging cell lines for propagating such vectors or viral vectors andto the processes for producing them.

All patents, patent applications, patent publications, scientificarticles, and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

Virus and nucleic acid vectors provide a means to deliver nucleic acidsequences to cells, and they are widely used in gene therapyapplications. Critical to effective gene therapy is the ability toestablish efficient expression of an Exogenous Nucleic Acid(s) in thetarget cell. Expression of exogenous nucleic acid in target cells cantake place when the Exogenous Nucleic Acid(s) is/are either in anintegrated or in an episomal state. Although expression in the episomalstate can take place in target cells, expression in most cases persistsfor only limited periods of time. In contrast, the expression ofExogenous Nucleic Acids in an integrated state can be maintained formuch longer periods.

Certain viruses have been of particular interest for use as vectors ingene therapy because of their ability to efficiently transfer and/orestablish stable expression of Exogenous Nucleic Acid in the targetcell. Although each particular family of virus may possess elements thatconfer specific advantages for development into a virus vector, eachvirus family also contains inherent features that limit its use as aviable means of human gene transfer.

Retroviruses have been a focus for development into virus vectorsbecause they can establish stable integration of viral sequences.Current retroviral vectors can be produced from packaging cells in whichthe gag, pol and env elements are provided in trans through a plasmid ormutated virus. These vectors can transduce sequences of up to 7.5 to 8.0kilobases. Nevertheless, several intrinsic features of retroviruses havehindered their use as virus vectors, and efforts to modify them toproduce safe and efficient vectors have led to low yields of virusvector or to the inefficient expression of the exogenous gene in thetarget cell. [Morgenstern, J. P. and Land, Hartmut Methods in MolecularBiology, Vol. 7: Gene Transfer and Expression Protocols, 1991, editedby: E. J. Murray The Humana Press Inc., Clifton, N.J.; Anderson, W FScience 256:808-813 (1992); Mulligan, R C Science 260:926-932 (19931];Smith, A E, Ann Rev. Microbiol. 49:807-838: Muzyczka, N., Curr. Top.Microbiol. Immunol. 158:97-129 (1992); Kotin, R. M., Human Gene Ther.5:793-801 (1994); and Berliner, K. L., Curr. Top. Microbiol. Immunol.158:39-66 (1992)). The contents of the foregoing book and publicationsare incorporated herein by reference. For example, it has beendemonstrated that in retrovirus vectors the level of expression directedby an internal promoter/enhancer can be suppressed up to 50-fold by theflanking LTRs, presumably as a result of interference betweentranscriptional regulatory units. [Methods in Molecular Biology, Vol. 7:Gene Transfer and Expression Protocols Edited by: E. J. Murray TheHumana Press, Inc. Clifton, N.J. (1991), supra; Emerman, M. and Temin,H. M., Cell 39:459-467 (1984); Emerman, M. and Temin, H. M., Mol. Cell.Bio. 6:792-800 (1986); Emerman, M. and Temin, H. M., Nucleic Acids Res.14:9381-9396 (1986)]. The foregoing book and publications are alsoincorporated herein by reference. Attempts to overcome this suppressionand achieve maximum expression of the exogenous nucleic acid have beenmade by deletion of the promoter and enhancer sequences within the U3region of the 3′ LTR in the provirus [Yu, S. F. et al. Proc. Natl. Acad.Sci. USA 83:3194-3198 (1986); Hawley, R. G. et al. Proc. Natl. Acad.Sci. USA 84:2406-2410 (1987); Yee, J. K. et al. Proc. Natl. Acad. Sci.USA 84:5197-5201 (1987)]. All of the foregoing publications areincorporated by reference into this application. Because the U3 regioncontains a polyadenylation signal, any deletions within this region caneliminate processing of nascent mRNA. In the absence of 3′ RNAprocessing, such as polyadenylation, newly transcribed mRNA is highlyunstable and, therefore, subject to immediate degradation. This accountsfor the observation that provirus mRNA was not detectable in a packagingcell line transfected with retrovirus DNA possessing such a deletion(Dougherty, J. P. and Temin, H. M., Proc. Natl. Acad. Sci. USA84:1197-1201 [1987], incorporated herein by reference). Addition of anexogenous SV40 polyadenylation signal to a site downstream from the 3′LTR has been used in an attempt to increase the virus mRNA level in thepackaging cells. Several problems arise from the use of this method. Theexogenous polyadenylation signal results in a lengthened viral mRNA withadditional U5 and SV40 polyadenylation signal sequences which are notpresent in the retrovirus vector RNA in the packaging cells and in thetarget cells. This extra sequence can not only sterically hinder boththe intermolecular and intramolecular transfer of templates duringreverse transcription of the viral vector RNA, but can also decrease thepackaging efficiency and the size of the exogenous nucleic acid sequencewhich can be inserted into the virus vector due to the size restrictionof the RNA which can be packaged (Whitcomb, J. M. and Hughes, S. H.[1992] Ann. Rev. Cell Biol. 8:275-306, incorporated herein byreference). In cases where reverse transcription does occur, theexogenous polyadenylation signal is lost during the process of reversetranscription and it cannot be used for polyadenylation of mRNAtranscribed from an internal gene which does not contain its ownpolyadenylation signal.

Virus vectors such as retroviruses that can randomly integrate into acell genome have the potential to disrupt the structure and function ofcell genes. The transcriptional elements within such a randomlyintegrated virus vector can activate potentially harmful genes such asoncogenes or genes triggering programmed cell death [Jaenisch, R.,Harbers, K, Schnieke, A et al., Cell 32:209-216 (1983); Fung, Y. T. etal., Proc. Natl. Acad. Sci. USA 78:3412-3422 (1981); Neel, B. G. et al.;Cell 23:323-334 (1981); Payne, G. S. et al. Cell 23:311-322 (1983);Lewin, B., Genes V, Oxford University Press, New York (1994)]. Thelast-mentioned book and the foregoing publications are incorporatedherein by reference. While removal of the transcriptional activity ofthe LTRs can reduce or eliminate the risk of unwanted gene activation bythe integrated virus vector, the promoters/enhancers of the exogenousnucleic acid can still act to activate cellular genes near the site ofintegration.

Whereas certain viruses possess useful properties for gene transfer,their use is limited by the requirement for a helper virus or by aninability to provide for stable transfer of Exogenous Nucleic Acid to atarget cell. For example, certain defective viruses can be propagated inpackaging cells that provide the required packaging components but withthe requirement for use of a helper virus. In order to insure safe useof such a virus vector preparation, however, the contaminating helpervirus must be removed and the virus vector product must be extensivelysafety tested for the presence of any contaminating helper virus. Thepresent invention overcomes these limitations by providing compositionsfor virus metamorphosis which can be used for propagation of virusvectors without the requirement of a helper virus.

The ability of a virus vector to integrate into the host genome providesdistinct advantages for establishing stable expression of ExogenousNucleic Acid in a target cell. The ability to integrate at specificsites is of further advantage by providing for a reduced possibility foran integrated vector to alter the structure and function of cellulargenes. Unlike integrating viruses such as retroviruses, however,adeno-associated virus (AAV) is a virus that has been demonstrated to beable to integrate into a specific region of a cell genome, namely theq13-ter region of human chromosome 19 [Samulski, R. J. et al. EMBOJournal (1991); Kotin, R. M. et al., Genomics 10:831-834 (1991), thecontents of both publications incorporated herein by reference]. Thisspecific integration is directed by the AAV inverted terminal repeatsand the Rep function [(Kotin et al., Proc. Natl. Acad. Sci. USA87:2211-2215 (1990), incorporated herein by reference]. While suchspecific integration makes AAV an attractive candidate for use as avirus vector, existing AAV vectors cannot integrate at specific sites ina target cell genome. Other features that hinder the use of AAV vectorsfor gene therapy are the size restriction of the internal gene, thedifficulty in growing virus in large amounts and the risk ofhelper-virus free contamination, all of which stem from the intrinsicmechanism of AAV replication.

By incorporating from different viruses the viral elements that mediatereplication, virus vectors that derive specific advantages from eachvirus can be created to overcome the limitations associated with eachvirus vector. For example, the transfer of site-specific integrationfunction from AAV into other virus vector systems can provide for suchproperties in a virus vector that may have useful properties for genetransfer but lacking any ability to integrate.

For gene delivery purposes, a virus vector can be developed from a virusthat is native to a target cell or from a virus that is non-native to atarget cell. In general, it is desirable to use a non-native virusvector rather than a native virus vector. While native virus vectors maypossess a natural affinity for target cells, such viruses pose a greaterhazard since they possess a greater potential for propagation in targetcells. In this regard, animal virus vectors, wherein they are notnaturally designed for propagation in human cells, can be useful forgene delivery to human cells. In order to obtain sufficient yields ofsuch animal virus vectors for use in gene delivery, however, it isnecessary to carry out production in a native animal packaging cell.Virus vectors produced in this way, however, normally lack anycomponents either as part of the envelope or as part of the capsid thatcan provide tropism for human cells. For example, current practices forthe production of non-human virus vectors, such as ecotropic mouse(murine) retroviruses like MMLV, are produced in a mouse packaging cellline. Another component required for human cell tropism must beprovided.

While non-viral nucleic acid complexes can provide significantadvantages for gene delivery, these advantages have not or cannot berealized by the use of non-viral nucleic acid complexes that rely onnon-specific binding components. The present invention overcomes theselimitations by providing for specific complex formation between nucleicacid and protein components wherein the binding of protein moleculesthat provide useful properties for gene transfer can be localized todefined regions of the nucleic acid construct. Such localization ofspecific binding proteins in the nucleic acid constructs can reduce oreliminate any interference with the region segments in the constructsthat are involved in or provide for biological activity. The presentinvention also provides for the controlled displacement of such specificbinding proteins from their cognate binding sites wherein suchdisplacement can remove any possible interference with biologicalfunction or can release proteins that can provide useful function in thecell.

SUMMARY OF THE INVENTION

The present invention provides novel vectors and viral vectors for usein systems for delivering and expressing desired genes and genesequences. One such novel vector is shown to be capable of expressing anexogenous gene or exogenous nucleic acid sequences in a target cell ofinterest. The vector comprises a viral vector, a viral vector nucleicacid, or a nucleic acid construct that comprises a viral vector nucleicacid sequence. The vector comprises the following nucleic acid componentor components: i) one or more native promoter/enhancer regions in whichat least one sequence segment has been modified, (ii) one or morenon-native promoter/enhancers or a non-native promoter's gene or genesegment, and (iii) a native viral vector terminator or a processingsignal or segment thereof, or both.

The present invention also provides a novel viral vector comprising avirus or viral portion having at least two adsorbing components on thesurfaces or envelopes thereof. One adsorbing component is directed to apackaging cell line for the vector, and the other adsorbing component isfor adsorbing to a target cell for delivering the vector.

Further provided by this invention is a novel viral vector comprising avirus or viral portion thereof in which at least two components on thesurfaces or envelopes are found. The first component is native to thevirus while the second component is generally characterized as beingnon-native to the viral vector, and further, being capable of adsorptionto a target cell of interest, while being incapable of adsorption to acell native for the same viral vector.

The present invention provides yet further a novel vector selected fromthe following group: a (i) viral vector, (ii) a viral nucleic acid, and(iii) a nucleic acid construct. The vector comprises a non-nativenucleic acid sequence coding for a segment, the segment being capable ofintegrating into a target cell's genome, and the vector itself beingcapable of producing or introducing a first nucleic acid in the targetcell. With respect to the first nucleic acid, it is itself capable ofproducing a second nucleic acid that comprises a portion of the firstnucleic acid. The second nucleic acid comprises the integration segmentand is itself capable of expressing an exogenous gene or an exogenousnucleic acid sequence as the case may be.

Also provided by this invention is a novel first vector selected fromthe group consisting of (i) a viral vector comprising a viral nucleicacid and a viral vector packaging component or components, (ii) a viralnucleic acid, and (iii) a nucleic acid construct. When introduced into apackaging cell, the first vector is capable of producing a second vectorselected from the group consisting of (a) a second viral vector, b) aviral nucleic acid, and (c) a second nucleic acid construct, each ofwhich group members are capable of expressing an exogenous gene orexogenous nucleic acid sequence in a target cell of interest. The firstvector is capable of producing the second vector in the packaging cell,and the packaging cell is capable of providing one or more packagingcomponents for the second viral nucleic acid. In this unique vector, thesecond viral nucleic acid or the second nucleic acid construct isstructurally different from the first (i) viral nucleic acid or thefirst (iii) nucleic acid construct. Alternatively, more than onepackaging component for the second viral vector may be different fromthe first viral vector packaging component or components (ii). As afurther alternative, both kinds or sets of structural differences may bepresent in the same vector. That is to say, the second viral nucleicacid or the second nucleic acid construct may be different from thefirst, and/or the packaging components for the second may be differentfrom the first.

This invention is also directed to novel packaging cell lines forpropagating any of the foregoing vectors or viral vectors, including thelast-mentioned first vector. Thus, the packaging cell line of thepresent invention provides at least two packaging components for thesurface or envelope of the viral vector. Other packaging cell lines forpropagating other viral vectors are also provided. In these, the cellline is non-native to the viral vector component or components butnative to the viral vector nucleic acid. The packaging cell lineexpresses one or more adsorbing components on its membrane or surface.Such adsorbing components are for adsorption to the non-native componentof the vector and broadly comprise receptor(s) or binding partner(s).

Processes for producing any of the novel viral vectors or viral vectornucleic acid of this invention are also contemplated and provided inthis disclosure. In these processes, the desired vector is introducedinto an appropriate packaging cell under conditions sufficient orappropriate to produce the viral vector or viral vector nucleic acid.

Still yet provided by this invention are novel and unique packaging celllines for propagating viral vectors independent of helper viruses. Insuch packaging cell lines, the viral vector comprises a nucleic acidcomponent and a non-nucleic acid component. The sequence or sequencesfor the viral vector nucleic acid component is stably integrated in thegenome of the cell line. The sequence or sequences for the non-nucleicacid component of the viral vector are introduced into the packagingcell line by various means. These means can involve transientexpression, episomal expression, stable integration expression, or anycombination of such foregoing means.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the general replacement strategy to a retroviral vectorsequence present in the plasmid pENZ1.

FIG. 2 shows the wild type 3′ LTR native sequence that was removed fromthe plasmid pENZ1 as well as the non-native modified 3′ LTR sequencethat replaced it (SEQ ID NO:15 and 16). The modified sequences aredesignated by bold italics.

FIG. 3 depicts the general replacement strategy for a retroviral vectorhaving an inactivated promoter/enhancer and a non-native polyadenylationsignal (the mouse histone H2A614 gene).

FIG. 4 depicts the general replacement strategy for a heterologousretroviral vector in which a polyadenylation processing signal from ahuman gene (G-CSF) with the AATAAA and mRNA destabilization elementsremoved is used to replace a region of the 3′ U3 snRNA.

FIG. 5 depicts the general replacement strategy for constructing aheterologous retroviral vector for delivering an exogenous nucleic acidthat transcribes a chimeric molecule composed of antisense RNA and rRNA.

FIG. 6 also depicts the general replacement strategy for constructing aheterologous retroviral vector for delivering an exogenous nucleic acidthat transcribes a chimeric molecule composed of antisense RNA and rRNA.

FIG. 7 also depicts the general replacement strategy for constructing aheterologous retroviral vector for delivering an exogenous nucleic acidthat transcribes a chimeric molecule composed of antisense RNA and rRNA.

FIG. 8 illustrates the construction of a retroviral vector DNA constructthat contains two adeno-associated virus (AAV) ITR sequences whereby onesequence is inserted into a site immediately downstream from the primerbinding site and the other sequence is inserted into a site justupstream from the retrovirus origin for second strand synthesis (ppt).

FIG. 9 illustrates the construction of a retroviral vector DNA constructcontaining two AAV ITR sequences whereby one sequence is inserted into asite immediately downstream from the primer binding site and the othersequence is inserted into a site from which the ppt sequences have beendeleted.

FIG. 10 illustrates the construction of a heterologous vector(retrovirus vector) DNA construct containing two AAV ITR sequences thatflank the primer binding site (PBS).

FIG. 11 illustrates the construction of a heterologous vector(retrovirus vector) containing two AAV ITR sequences whereby onesequence is inserted into a site immediately downstream from the primerbinding site and the other sequence is inserted into a site justupstream from the retroviral origin for second strand DNA synthesis(ppt).

FIG. 12 illustrates the construction of a heterologous vector(retrovirus vector) containing two AAV ITR sequences whereby onesequence is inserted into a site immediately downstream from the primerbinding site and the other sequence is used to replace the originalretroviral sequences for second strand DNA synthesis (ppt).

FIG. 13 illustrates the construction of a heterologous vector(retrovirus vector) containing two AAV ITR sequences that flank theprimer binding site (PBS). The ppt sequences are removed and the AAV repsequences are inserted in their place.

DETAILED DESCRIPTION OF THE INVENTION

The definitions below are useful to an understanding of the presentinvention and this disclosure.

DEFINITIONS

Heterologous Vector: A virus vector or non-virus vector, includingnon-viral specific complex that consists of at least one Non-NativeVector Component and that is capable of delivering an Exogenous NucleicAcid to a cell and which can facilitate Exogenous Nucleic Acidexpression in a cell. The Heterologous Vector contains a functionalnative segment or segments that interfere with the expression of anexogenous gene or an exogenous nucleic acid. The native segment orsegments have been modified wherein the interference is reduced oreliminated and/or native termination/RNA processing is retained.

Non-Native Vector Component: A nucleic acid sequence derived from anybiological system, or an altered or modified native element, that formsa components) of a Heterologous Vector. The components) function in ormediate directly or indirectly in a cis or trans fashion either in vivoor in vitro to provide or effectuate 1) expression (includingtermination signal) of the Exogenous Gene or Exogenous Nucleic Acid ofthe Heterologous Vector in a target cell of interest; 2) integration;and 3) propagation, yield and assembly.

Expression Cassette (or the expression of exogenous nucleic acidsequence or exogenous gene): A nucleic acid sequence that contains allthe elements required for exogenous gene expression or the expression ofan exogenous nucleic acid sequence or segment, and that is inserted intoa vector for the purpose of expression in a target cell of interest.Such elements embrace both native and non-native vector components orcombinations thereof, including modified or unmodified promoter/enhancersequences for the expression of Exogenous Nucleic Acid or Exogenous Genethat may contain a gene or gene segment corresponding to the non-nativepromoter/enhancer, modified or native viral promoter/enhancers andsignals for termination, RNA processing, polyadenylation and RNAtransport.

cis effect: The effect exerted by one functional segment of a vectornucleic acid on the function of another distal sequence of vectornucleic acid.

Heterologous Virus Vectors

The present invention provides compositions and methods of use forHeterologous Vectors that have useful properties for gene delivery tocells, i.e., 1) efficient propagation in a packaging cell and 2) thesafe and efficient expression of Exogenous Nucleic Acid in a cell. Thesebenefits are achieved by the use of Non-Native Vector Components thatcan provide one or more such properties to a virus vector.

Expression of Exogenous Nucleic Acid in a virus vector can in many casesbe inefficient because of the virus vector native promoters/enhancersactivity that interferes: with the function of non-nativepromoters/enhancers driving Exogenous Nucleic Acid expression (Emmermanand Temin, 1986, contents of which are incorporated herein byreference). Efforts to eliminate this interference by deletion of thevirus vector native promoters/enhancers produce cis effects that occurat sites distal to the modification site. Such cis effects may lead toloss or reduction in termination and/or RNA processing which causesreduction or a diminishment in the expression of Exogenous Nucleic Acidas well as greatly reduced or an altogether eliminated ability topropagate efficiently in a packaging cell. The addition of a non-nativepolyadenylation signal to a site downstream from the 3′ LTR has beenused in an attempt to restore the lost function (Dougherty and Temin,1987, incorporated herein by reference). Such an approach is limited inseveral critical aspects. This exogenous polyadenylation signal resultsin a lengthened viral mRNA with additional U5 and SV40 polyadenylationsignal sequences which are not present in the retrovirus vector RNA inthe packaging cells and in the target cells. This extra sequence can notonly sterically hinder both the intermolecular and intramoleculartransfer of templates during reverse transcription of the viral vectorRNA, but it can also decrease the packaging efficiency and the size ofthe exogenous nucleic acid sequence which can be inserted into the virusvector due to the size restriction of the RNA which can be packaged(Whitcomb and Hughes, 1992, incorporated herein by reference). In caseswhere reverse transcription does occur, the exogenous polyadenylationsignal is lost during the process of reverse transcription and it cannotbe used for polyadenylation of mRNA transcribed from an exogenous genewhich does not contain its own polyadenylation signal.

It is another aspect of this invention to overcome the above limitationsin the art by providing modification in the natural promoters/enhancerssegment through a variety of means including substitution, addition,mutation or any combination thereof. The present invention overcomesthese limitations by providing in one feature the artificialreconstitution of the native promoters/enhancers segment of the vectorwhich has been demonstrated to reduce or eliminate such cis effects inthe vector. This reconstitution or modification is carried out inaccordance with this invention, for example, through the replacement ofHeterologous Vector nucleic acid sequences with Non-Native VectorComponents that can provide such restoration or even improvement ofvector virus functions. This reconstitution can be accomplished byreplacement of virus vector promoter and/or enhancer sequences withNon-Native Vector Components to provide a virus vector with an mutatedLTR in which the native promoter/enhancer function is inactivated insuch a manner that eliminates interference with non-nativepromoter/enhancer functions. In this case, the virus vector retainsfully active native termination functions and native RNA processingfunctions for expression of virus vector RNA in a packaging cell and forexpression of Exogenous Nucleic Acid in a target cell.

Thus, the present invention provides a vector comprising a viral vector,a viral vector nucleic acid, or a nucleic acid construct that comprisesa viral vector nucleic acid sequence. The vector is capable ofexpressing an exogenous gene or exogenous nucleic acid sequences in atarget cell of interest, the vector comprising a nucleic acid componentor components. The latter nucleic acid component or components comprise(i) one or more native promoter/enhancer regions in which at least onesequence segment has been modified, (ii) one or more non-nativepromoter/enhancers or a non-native promoter's gene or gene segment, and(iii) a native viral vector terminator or a processing signal or segmentthereof, or both. Additionally, the aforementioned viral vector furthercomprises a non-native terminator or two or more modified sequencesegments.

Such modifications may take various forms. For example, a nativesequence segment can be substituted by a non-native sequence segment inthe one or more promoter/enhancer regions of the vector. Further, thesubstitution can be of approximately the same size. In another aspect,the modification can comprise a mutation selected from any of the groupmembers represented by a point mutation, a deletion, an insertion, and asubstitution, or a combination of any of the foregoing.

In one preferred aspect, the viral vector is a retrovirus. In another,the terminator, or processing signal, or both, as the case may be, caninclude a polyadenylation signal. In addition, such a viral vector cancomprise a segment of the viral vector terminator or a segment of theprocessing signal, or both. Additionally, the function of the one ormore promoter/enhancers will have been reduced, inhibited or eliminatedin the present viral vector.

With respect to the one or more non-native promoters, these are capableof producing an RNA lacking a polyadenylation signal. A number ofnon-native promoters can be used in accordance with this invention.Simply by way of example, such non-native promoters can be selected fromthe group of genes represented by or designated as snRNA, tRNA, andrRNA, or a combination of any of the foregoing.

In another aspect of this invention, the afore-described viral vectorfurther comprises one or more gene or gene segment sequences of thesnRNA, tRNA or rRNA gene or genes. The snRNAs are well described in theliterature, and these include, for example, any of the members selectedfrom the group consisting of U1, U2, U3, U4, U5, U6, U7, U8, U9, U10 andU11, or a combination of any of the foregoing.

It should also be pointed out that in the viral vector described above,one or more non-native promoters gene or gene segment sequence can orwill have been modified. Such modifications can also take a number offorms, including the substitution or replacement of or addition to theone or more non-native promoter's gene sequence with the exogenous geneor an exogenous nucleic acid sequence.

Non-Native Vector Components useful for these purposes includenon-native nucleic acid sequences in the vector. Such nucleic acidsequences can be derived from any biological system or can be chemicallysynthesized or can be prepared by recombinant DNA methods or by anycombination of such methods, Such sequences can be approximately thesame size as the vector virus sequences that are replaced. Thus, suchsequences can range in size from approximately 2 to approximately 188bases or base pairs in length, or longer. Such sequences can be used toreplace one or more sequences in such regions of the virus vector aspromoter and/or enhancer sequences, or any other native sequences inwhich its ability leads to cis effects. Such replacements can be carriedout by the conventional methods of recombinant DNA (see Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning, 2nd ed. Cold SpringLaboratory, Cold Spring Harbor, N.Y., 1989, the contents of whichtextbook are incorporated herein by reference), and they can beconveniently performed on virus vector nucleic acid genomes or fragmentsthereof that are present as double stranded DNA in plasmids.

Modifications to provide reconstitution of cis effects in such vectorsas retroviruses can, for example, be accomplished by replacements in theU3 region of the 3′ LTR region of a retrovirus vector genome present ina plasmid, i.e., a vector nucleic acid construct. The propagation ofretrovirus vectors can proceed by introduction of such a plasmid into apackaging cell wherein transcripts of the virus vector genome areproduced and reverse transcribed after transduction into a target cell.As a result of these processes, modifications made to the 3′ LTR in avector plasmid will be present in the 5′ LTR of propagated retrovirusvectors.

Reconstitution has been accomplished according to the teachings of thisinvention in a retrovirus vector provirus DNA contained in a vectorplasmid (designated pENZ-1) by modifications of the 3′ LTR. Threeseparate sequences from the promoter/enhancer region were replaced withnon-native sequences of approximately the same size. A sequence of 188base pairs from the enhancer region of the 3′ LTR was replaced with anunrelated sequence of 188 base pairs derived from the bacterial neogene. Two separate sequences in the promoter region, one of 2 bases andthe other of 6 bases, were also replaced with nucleic acid segments ofthe same size. Introduction of this provirus DNA construct into apackaging cell (either GP+E-86 or PA 317) produced retrovirus vectors attiters of up to 10⁶ as measured by transduction of G418 resistance. Thisis illustrated in FIGS. 1 and 2.

Thus, the present invention also provides a first vector selected fromthe group consisting of (i) a viral vector comprising a viral nucleicacid and a viral vector packaging component or components, (ii) a viralnucleic acid, and (iii) a nucleic acid construct, wherein whenintroduced into a packaging cell, the first vector is capable ofproducing a second vector selected from the group consisting of (a) asecond viral vector, (b) a viral nucleic acid, and (c) a second nucleicacid construct, each being capable of expressing an exogenous gene orexogenous nucleic acid sequence in a target cell of interest. The firstvector is capable of producing in the packaging cell the second vector,and the packaging cell is capable of providing one or more packagingcomponents for the second viral nucleic acid. The second viral nucleicacid or the second nucleic acid construct is structurally different fromthe first (i) viral nucleic acid or the first (iii) nucleic acidconstruct, or more than one packaging component for the second viralvector is different from the first viral vector packaging component orcomponents (ii), or both instances of structural differences may bepresent in this first vector. In one aspect, the first vector comprisesa retrovirus and the second vector comprises adeno-associated virus(AAV).

With respect to these aforementioned structural differences, thesecomprise or take on any number of forms, including any differences thatare selected from the following group members: the nucleic acid chemicalnature, the nucleic acid form, the nucleic acid size, and functionalelements, or a combination of any of the foregoing. With respect to thenucleic acid chemical nature, the second viral nucleic acid or thesecond nucleic acid is selected from any of the group members consistingof or designated as RNA and DNA, and the (i) viral nucleic acid or the(iii) nucleic acid construct comprises a different member of the groupto impart a structural difference between the elements. With respect tothe nucleic acid form, the second viral nucleic acid or the secondnucleic acid is selected from any of the group members consisting ofsingle-stranded, double-stranded and partially double-stranded, and the(i) viral nucleic acid or the (iii) nucleic acid construct comprises adifferent member of the group to impart a structural differencetherebetween. With respect to the nucleic acid size, the second viralnucleic acid or the second nucleic acid comprises a segment of the (i)viral nucleic acid or the (iii) nucleic acid construct. With respect tothe functional elements, the second viral nucleic acid or the secondnucleic acid comprises one or more promoters, one or more enhancerregions, an integration segment and a terminator, or a portion or asegment or a combination of any of the foregoing, and the (i) viralnucleic acid or the (iii) nucleic acid construct comprises a differentmember of the group to impart a structural difference therebetween.

In preferred aspects of this invention, the first vector comprises aretrovirus and the second vector comprises adeno-associated virus. Inother preferred aspects, the first vector comprises adeno-associatedvirus and the second vector comprises a retrovirus.

Retrovirus vectors can also be reconstituted with a nucleic acidsequence for a non-native promoter.

In comparison to a non-reconstituted modified retroviral vector that hasexclusively lost its propagation capability, the reconstitution of ciseffects, as described earlier, can thus provide virus vectors with a)the ability to express Exogenous Nucleic Acid at a maximum level, and/orb) the ability to propagate efficiently in a packaging cell.Furthermore, as a result of the inactivation of virus vectorpromoter/enhancer function, such Heterologous Vector viruses could haveproperties for safe use in a gene delivery system. For example, wheresaid Heterologous Vector contains a non-native promoter which cannotdirect the transcript of a polyadenylated RNA, then such a vector has agreatly reduced or lost ability to activate the expression ofpolyadenylated mRNA or non-polyadenylated mRNA from cellular genes thatutilize either polymerase I-dependent, polymerase II-dependent orpolymerase III-dependent promoters as a result of random integration ofthe vector. The present invention provides additional compositions forthe safe use of Heterologous Vectors by the use of non-nativepromoters/enhancers for the expression of Exogenous Nucleic Acid whereinsuch promoters/enhancers lack the ability to provide poly(A) signalsequence for activating expression of polyadenylated mRNA. The use ofsuch non-native promoters/enhancers for expression of Exogenous NucleicAcid in such Heterologous Vectors can provide safe virus vectors inwhich the ability to activate polyadenylated mRNA synthesis of cellulargenes by either virus vector native promoters/enhancers or by non-nativepromoters/enhancers is markedly reduced or eliminated.

Non-native elements in the vector that provide safe expression ofExogenous Nucleic Acid can be derived from any biological system and caninclude promoters/enhancers and compatible processing signals that donot direct the transcription of polyadenylated RNA. Thesepromoters/enhancers include such elements that are recognized by RNApolymerase I, RNA polymerase II or RNA polymerase III that provide forthe synthesis of cellular RNA elements such as ribosomal RNA, transferRNAs, small nuclear RNAs such as U1, U2, U3, U4, U5, U6, U7, U8, U9. U10and U11. These sequences of cellular RNA elements can be useful to thepractice of this invention wherein they can be used to form chimeric RNAmolecules with RNA sequences that can provide such biological functionsas antisense regulation of gene expression, ribozyme activity, sensesequences for expression of an exogenous gene or exogenous nucleic acid,and the like.

Such sequences providing for biological activity can be incorporatedinto such a cellular RNA gene or gene segments by partial or completereplacement of some or all of the sequences of the cellular RNA gene orgene segments, or by the addition of such sequences to the RNA genesequence. Using the methods of recombinant DNA, such chimeric moleculescan be formed by utilizing the cloned sequences for the cellular RNAelements and coding sequence for RNA transcripts of exogenous gene orgene segments or exogenous nucleic acid. Such a chimera could be all orin part chemically synthesized. Nucleic acid sequences providing for thesynthesis of such chimeric RNA molecules can be convenientlyincorporated into double stranded Heterologous Vector DNA present in aplasmid by the methods of recombinant DNA as described above.Heterologous Vectors providing for expression of such RNA compositionsare presented in FIGS. 5, 6 and 7.

For the use of such chimeras formed between a cellular RNA gene or genesegment and a sequence coding for an exogenous gene or exogenous nucleicacid, modifications of the cellular RNA gene may lead to enhancedfunction of the cassette. For example, in chimeric molecules formedbetween an antisense RNA and a U1snRNA, such modifications of the U1snRNA that could provide a U1 snRNP complex with loss of catalyticfunction and/or loss of transport properties and/or loss of proteininteraction could further the expression of antisense function in acell. Modifications that could provide such properties could result fromdeletions, additions or from alterations of one or more bases of thesequence of such a molecule. Such a modified U1 system has been preparedfrom a U1 molecule containing an altered base sequence, i.e., a changeof C to T at position 4 of the U1 RNA transcript present in plasmidpHSD-4 (Manser, T. and Gesteland, R., Cell 29:257-264 (1982), thecontents of which are incorporated by reference). This U1 was used toform a chimeric molecules with each of three different antisensesequences, each directed against an HIV-1 target sequence. Replacementwas done by replacement of a portion of the transcribed region of U1(present in pHSD-4) with each antisense sequence.

Such chimeric molecules can be inserted into Heterologous Vectors suchas retroviruses to provide for delivery to and expression of suchchimeric antisense molecules in a target cell. The absence of any vectorpromoter activity and the lack of production of any peptide by the virusvector sequences or by the Exogenous Nucleic Acid thus produces nonon-native protein in a target cell, thus eliminating the possibilityfor an immune response. See Roy-Chowdhury et al., “Novel ProcessesImplementing Selective Immune Down Regulation (SIDR),” U.S. patentapplication Ser. No. 08/808,629 filed on Feb. 28, 1997, the contents ofwhich are incorporated herein by reference).

Such immunogenically silent vectors could be especially useful for exvivo gene transfer to such cells as hematopoietic stem cells. Treatmentof virus infections of these cells could benefit from the use of thesevectors for the delivery of biologically active RNAs such as antisense(including ribozymes) and sense RNAs directed against the infectingviruses. For example, treatment of diseases such as HIV-1 (and certainviral leukemias) could benefit from gene transfer of such sequences tohematopoietic stem cells as a means for providing a source of CD4+ cellswith resistance to HIV-1 infection. Cells for this purpose could bederived from autologous sources such as the bone marrow or thecirculating cells of the donor or from heterologous sources such asfetal cord blood. Such cells could be grown and transduced with such avector according to procedures such as described by (Nolta et at. 1995Blood 86.101-110; Xu et al., Blood 86:141-146 (1995); Bertolini et al.,Cancer Research 56:2566-2572 (1996); and Wells et al., Gene Therapy2:512-520 (1995)). The contents of the foregoing publications areincorporated herein by reference. These procedures could utilizeautologous sera and stroma. Cells so treated could be administered topatients who had previously undergone partial or complete ablation.

Immunogenically silent Heterologous Vectors constituted as describedabove could also be utilized for in vivo gene delivery. Such tissues asliver, lung, kidney, brain, muscle, epithelial tissues and other tissuesnot easily amenable to ex vivo procedures could be targeted by theadministration of such virus vectors to the bloodstream or by directinjection into a tissue or organ. Such treatment procedures can be usedin combination with other methods.

Virus Metamorphosis

The present invention provides compositions and methods of use forvectors or viral vectors that can, in an appropriately constructedpackaging cell or in a target cell, propagate to a virus vector nucleicacid or virus vector which differ from the original vector in variouselements as described herein, or even a second virus vector, or producea virus vector genome of a different or even second virus vector orproduce a nucleic acid that substantially resembles the genome of adifferent or second virus vector. Such a process of virus metamorphosiscan be mediated by a virus vector modified in its nucleic acid sequenceby the incorporation of one or more non-native sequences. Introductionof such a modified nucleic acid component or components into anappropriately constructed packaging cell can provide propagation of suchsecond virus vectors from the original vector, or its introduction intoa target cell can produce a nucleic acid of a second virus vector orvirus vector nucleic acid which contains properties other than found inthe original vector, including integration into the target cell genome.Compositions are also provided for packaging cells that can be modifiedto provide virus metamorphosis by the incorporation of componentsnon-native to said packaging cell. Compositions for virus metamorphosiscan be useful for the production of defective virus vectors wherein suchviruses can be propagated without the requirement for a helper virus andwhich can also be useful to provide properties for the integration ofExogenous Nucleic Acid into the genome of a target cell.

Whereas certain viruses possess useful properties for gene transfer,their use is limited by the requirement of a helper virus for virusvector production or by an inability to provide for stable transfer ofExogenous Nucleic Acid to a target cell or for integration of ExogenousNucleic Acid at preferred sites of a target cell genome. For example,certain defective viruses can be propagated in packaging cells thatprovide the required packaging components but with the requirement foruse of a helper virus. In order to insure safe use of such a virusvector preparation, however, the contaminating helper virus must beremoved and the virus vector product must be extensively safety testedfor the presence of any contaminating helper virus. The presentinvention overcomes these limitations by providing compositions forvirus metamorphosis which can be used for propagation of second virusvectors without the requirement for a helper virus.

Among the novel and useful viral vectors of the present invention is onecomprising a virus or viral portion having on a surface or an envelopethereof at least two adsorbing components, one component for adsorptionto a packaging cell line for the vector, and the other component foradsorption to a target cell for delivery of the vector. Bothaforementioned components can be native to the viral vector, or both canbe non-native to the viral vector, or in some instances one componentcan be native and the other component can be non-native. When at leastone component is native to the viral vector, one of the components canbe ecotropic or amphotropic. Such non-native components are known in theart and can take a number of forms. These include, by way of example,any of the members selected or derived from the group consisting ofHuman Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis CVirus (HCV), Herpes Simplex Virus (HSV), and Vesticular Stomatis Virus(VSV), and a part or portion thereof, or a combination of any of theforegoing. In the case of HIV or its part or portion thereof, thenon-native component can comprise gp120. In the case of HBV or HCV, thenon-native component comprises a surface antigen. One or the other orboth components of the viral vector can be selected from any of themembers of the group consisting of a protein, an oligo- or polypeptide,a glycoprotein, a fused peptide, a recombinant peptide, a modifiedprotein, or a combination of any of the foregoing.

In preferred aspects, the above described viral vector comprises aretrovirus such as a murine retrovirus.

Vectors that can provide for vector metamorphosis can also be used toprovide integration properties to the vectors derived from the originalvector. While certain viruses possess useful properties for genedelivery, their use is limited by an inability to integrate and/or toremain stably associated with a target cell, and such virus vectors canthus only provide for expression of Exogenous Nucleic Acid for a limitedperiod. Compositions for virus metamorphosis can be used to provide forstable expression of Exogenous Nucleic Acid by providing properties forstable integration into a target cell genome of the second vector wherethe original vector lacks integration capability. Such properties can beprovided to a vector or virus vector by components non-native to such avector or virus vector wherein such properties can be derived from otherviruses or from other biological systems or synthetically.

Virus metamorphosis can proceed in a packaging cell or in a target cellby the introduction into said cell of the nucleic acid of an initiatingvector (or first virus vector) wherein said nucleic acid is a componentof a virus vector, is a virus vector nucleic acid or is a nucleic acidconstruct or a component thereof. Propagation of said nucleic acid in apackaging cell or in a target cell can directly or indirectly yield anucleic acid with properties native to a virus vector (second virusvector) that is unrelated to the first virus vector wherein the nucleicacid of the second virus vector differs from the nucleic acid of thefirst virus vector in i) complexity, wherein it can be shorter orlonger, ii) in chemical nature wherein it can be either single or doublestranded RNA or DNA or partially single stranded and partially doublestranded RNA or DNA and iii) in the function of promoters/enhancers,integration sequences and termination, processing sequences, or thedifference lies in packaging surface component or components. Theproperties of the second vector nucleic acid could provide for thepackaging of the second vector in a packaging cell which is constructedto provide the required components for the second vector packaging.Alternatively, the nucleic acid of such an second virus vector soproduced in a target cell could contain properties for its incorporationinto the genome of said cell.

Virus vectors useful for the practice of this invention can be derivedfrom plant, bacterial, animal and human viruses wherein these can bemodified by components non-native to said initiating: vector virus. Suchcomponents can normally be derived from other viruses but could also bederived from other biological systems or made synthetically. Suchcomponents include but are not necessarily limited to nucleic acidsequences that provide for virus propagation, integration function andgene expression for virus components. These include the LTR sequences ofretroviruses, the integrase protein of retroviruses, the reversetranscriptase of retroviruses, the ITR sequences of AAV, the rep genesof AAV, the cap genes of AAV and other components that can provideuseful functions.

The nucleic acid sequences of virus vectors used to initiate virusmetamorphosis, i.e., and first virus vector, can be convenientlyconstructed by the methods of recombinant DNA wherein the non-nativevector components can be incorporated into a vector nucleic acidsequence.

Packaging cells for the practice of this invention can be prepared bythe introduction of nucleic acid sequences normally derived from boththe initiating vector and the second vector. Such nucleic acid sequencescan provide for the synthesis of the second vector component(s)including packaging components, polymerases or other required enzymes,and for the synthesis of the second vector nucleic acid. Such nucleicacid sequences can present in such cells in either an integrated or inan episomal state.

Virus vectors that can be utilized for virus metamorphosis includeretroviruses like the Moloney murine leukemia virus (MMLV). A retrovirusvector (vector, vector nucleic acid, or nucleic acid construct) can bemodified to propagate an second virus vector, such as the AAV, byincorporating a sequence of the AAV ITR into the retroviral vectornucleic acid sequence. Two such sequences can be inserted into theretrovirus vector nucleic acid. Such vector can direct the synthesis ofretrovirus vector RNA in a packaging cell and the packaging cell linecan provide reverse transcriptase for synthesis of AAV DNA. ExogenousNucleic Acid are present in the region flanked by the AAV ITRs. Theretroviral vector nucleic acid can be further modified by inactivationof the ppt sequence segment function (or others), thus eliminatingsynthesis of the second DNA strand after reverse transcription as ameans of providing single stranded DNA copies of the second vector (forexample, AAV). This can be accomplished by deletion of the ppt sequenceor by the replacement of the retroviral ppt sequence with one of the AAVITR sequences or with an AAV rep sequence by methods described elsewherein this patent. AAV rep and cap nucleic acid sequences can be providedto packaging cells as part of the retrovirus nucleic acid component orsuch sequences can be provided separately on plasmids or other nucleicacid entities either inserted into a cell genome or present in thepackaging cell in an episomal state or in a transient state. Thisinvention further provides a viral vector comprising a virus or viralportion thereof having on a surface or an envelope at least twocomponents, the first component being native to the virus, and thesecond component characterized by three characteristics. First, it isnon-native to said viral vector. Second, it is capable of adsorption toa target cell of interest. Third, the second component is incapable ofadsorption to a cell native for the viral vector. In a preferred aspect,the viral vectors is a retrovirus. Suitable or appropriate retroviruseshave been well characterized in the literature and can take a number ofdiverse forms. Merely by way of example, such retroviruses can beselected from any of the members of the group consisting of a murineleukemia virus, a human immunodeficiency virus, a human T cell leukemiavirus and a Gibbon ape leukemia virus, or a combination of any of theforegoing.

The non-native component in the above-described viral vector can alsotake a number of forms, all of which are well described in theliterature. These include any or all of the following members selectedor derived from the group consisting of Human Immunodeficiency Virus(HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Herpes SimplexVirus (HSV), and Vesticular Stomatis Virus (VSV), and a part or portionthereof, or a combination of any of the foregoing. In the case of HIV,the derived member can comprise gp120. In another instance, thenon-native component can comprise HBV or HCV surface antigen.

The present invention contemplates a number of useful target cells,including any of the members selected from the group consisting of Tcells, liver cells, bone marrow cells and epithelial cells, or acombination of any of the foregoing.

The present invention also provides a vector selected from the groupconsisting of a (i) viral vector, (ii) a viral nucleic acid, and (iii) anucleic acid construct, the vector comprising a non-native nucleic acidsequence coding for a segment, the segment being capable of integrationinto a target cell's genome, and the vector being capable of producingor introducing a first nucleic acid in the target cell, the firstnucleic acid being capable of producing a second nucleic acid thatcomprises a portion of the first nucleic acid, the second nucleic acidcomprising the integration segment and being capable of expressing anexogenous gene or an exogenous nucleic acid sequence. In one aspect,this vector can comprise a viral vector and the integration segment canbe non-native to the viral vector. The vector can also comprise a viralnucleic acid and the integration segment can also be non-native to theviral vector. In one preferred embodiment, the viral vector comprisesadenovirus. In another, the first nucleic acid comprises a retrovirusand the second nucleic acid comprises adeno-associated virus (AAV). Inyet another, the first nucleic acid comprises AAV and the second nucleicacid comprises a retrovirus. Still further, the second nucleic acidsequence comprises retroviral LTR or AAV.

This invention also provides a process for producing any of the viralvectors or viral vector nucleic acids as disclosed herein or claimedbelow. Such a process typically comprises the steps of providing suchvector and introducing it into a packaging cell under conditions toproduce the viral vector or said viral vector nucleic acid. In oneaspect, the nucleic acid construct can be been modified in apromoter/enhancer region, in a non-native promoter. In other aspects ofthe just described process, the nucleic acid construct is capable ofstable integration into the genome of said packaging cell line. Itshould not be overlooked that in the case where a nucleic acid constructis employed in such process, the construct can be modified by means ofan episome or by means of transient expression.

This invention also provides a packaging cell line for propagating aviral vector independent of a helper virus. The viral vector cancomprise a nucleic acid component and a non-nucleic acid component. Thesequence or sequences for the viral vector nucleic acid component can bestably integrated in the genome of the cell line, and the sequence orsequences for the non-nucleic acid component of the viral vector areintroduced into the packaging cell line by a means selected from thegroup consisting of transient expression, episomal expression, stablyintegrated expression, or a combination of any of the foregoing.

Packaging cells for this purpose can be prepared to contain retroviralreverse transcriptase sequences in order to provide for reversetranscription of transcripts produced from the initiating vector(retrovirus).

An example of a virus vector that provides for production of an secondvector by virus metamorphosis is presented in Examples 7, 8 and 9, andFIGS. 8, 9 and 10. AAV ITRs and sequences for AAV rep are inserted intoan MMLV retrovirus vector sequence contained in a vector construct. Apackaging cell is prepared from mouse 3T3 cells by the stable insertioninto said cell of retrovirus reverse transcriptase sequences in order toprovide for reverse transcription of retrovirus vector transcripts andsequences for AAV cap to provide for AAV virus vector packaging.Following introduction of the initiating retrovirus nucleic acidcomponent (present on a plasmid) into a into such a packaging cell,reverse transcription of the retrovirus vector genome yields a singlestranded retrovirus DNA containing two AAV ITR sequences that areseparated by approximately 4.5 kb wherein they flank an AAV rep gene anda sequence for Exogenous Nucleic Acid. Such DNA copies, by virtue of theAAV ITR sequences and the AAV rep function can be replicated to produceAAV vector sequences which contain the AAV rep sequences and theExogenous Nucleic Acid sequence. The presence of cap proteins and theAAV packaging signal provide for packaging of such AAV vector viruses.

This invention also provides a packaging cell line for propagating anyof the viral vectors of the present invention, as disclosed or claimedherein. Such packaging cell line can provide, for example, at least twopackaging components for the surface or envelope of the viral vector. Inthe packaging cell line, the cell line can be native to the viralvector. The viral vector itself can comprise in preferred aspects aretrovirus. The cell line for use in the packaging line of thisinvention, can take on a number of forms known in the art, including,for example, any of the members selected from the group consisting ofNIH 3T3, U937, H9 and 293, or a combination of any of the foregoing.

In other aspects, any sequences for both the surface or envelopecomponents in the packaging cell line are stably integrated in achromosome or chromosomes of the packaging cell line. Furthermore, asequence of a surface or envelope component can be stably integrated ina chromosome or chromosomes of the packaging cell line, and a sequenceof another surface or envelope component can be transiently expressed.Still yet further, a sequence of said envelope component can be stablyintegrated in a chromosome or chromosomes of said packaging cell line,and a sequence of the surface component is transiently expressed. Inother aspects, any sequence for both the surface or envelope componentsin the packaging cell line can be transiently expressed.

This invention also provides a packaging cell line for propagating otherviral vectors as disclosed or claimed herein. In such instances, thecell line can be non-native to the viral vector component or componentsbut native to the viral vector nucleic acid. The packaging cell lineexpresses on its membrane or its surface a receptor or receptors orbinding partner or partners for adsorption to the non-native componentfor the vector.

Virus metamorphosis can also provide for integration of ExogenousNucleic Acid into a cell genome. The ability of the second vectornucleic acid to integrate into the host genome provides distinctadvantages for establishing stable expression of Exogenous Nucleic Acidin a target cell. However, some viruses lack this property but possessother useful properties for gene delivery, such as affinity for certaincell types, stability in human or animal tissues, efficient delivery ofnucleic to target cells. The present invention provides compositions andmethods of use for virus vectors that, through compositions for virusmetamorphosis, can provide for the integration of Exogenous Nucleic Acidinto a target cell genome. Such compositions can also provide for suchintegration to occur at preferred sites in the target cell genome.

Virus vectors possessing integration properties can be constructed bythe incorporation of non-native components into the virus vector genome.Such useful components include such entities as certain nucleic acidsequences such as those containing integration signal sequences andcertain vector nucleic acid conformations such as secondary structure.Non-native components useful for these purposes include such nucleicacid sequences such as retrovirus LTRs, reverse transcriptase andintegrase that can provide for integration at random sites in a targetcell genome. Virus vectors can also be modified with non-nativecomponents that provide for integration at preferred sites in a targetcell. Such sequences include ITR sequences and the rep genes derivedfrom AAV. These can be provided to a variety of virus vectors, includingretrovirus.

Virus vectors containing non-native components that provide integrationat preferred sites can be constructed by the methods of recombinant DNAas described above. Virus vectors such as retroviruses vectors can bemodified for this purpose by the incorporation into the retrovirusnucleic acid component of two such ITR sequences wherein, followingreverse transcription of vector RNA in a cell, the ITR sequences willflank a sequence or sequences containing Exogenous Nucleic Acid.Sequences for AAV rep function can also be incorporated into such aretrovirus vector genome or these sequences can be provided on aseparate entity such as a virus or a nucleic acid construct such as aplasmid. The presence of the AAV ITR sequences in the double strandedproduct of reverse transcriptase and the AAV rep function in the cellcan provide the capability for site specific integration into the targetcell genome. This process is illustrated in FIGS. 10, 11 and 12. Theprocess described above can also be performed through the production ofa single stranded DNA product of the reverse transcriptase reaction.This can be achieved as described above by inactivation of the region ofthe retroviral genome involved in the initiation of second strand DNAsynthesis, i.e., the ppt sequence (FIGS. 11 and 12).

Multitropic Virus Vectors

Gene delivery systems are known in the art. See, for example, Rabbani etal., U.S. patent application Ser. No. 08/574,443, filed on Dec. 15,1995, contents of which are incorporated by reference herein.

For gene delivery purposes, a virus vector can be developed from a virusthat is native to a target cell or from a virus that is non native to atarget cell. In general it is desirable to use a non-native virus vectorrather than a native virus vector. While native virus vectors maypossess a natural affinity for target cells, such viruses pose a greaterhazard since they possess a potential for propagation in target cells.In this regard animal virus vectors, wherein they are not naturallydesigned for propagation in human cells, can be useful for gene deliveryto human cells. In order to obtain sufficient yields of such animalvirus vectors for use in gene delivery, however, it is necessary tocarry out such production in a native animal packaging cell. However,virus vectors produced in this way normally lack any components eitheras part of the envelope or as part of the capsid that can providetropism for human cells. For example, current practices for theproduction of non-human virus vectors, such as ecotropic mouseretroviruses like MMLV, are produced in a mouse packaging cell line.Although producing a high titter, this vector lacked affinity for thetarget human cell. Alternatively, amphitropic vectors could be used butthe titter could be much lower.

This invention overcomes this limitation in the prior art by providingcompositions and methods of use for novel virus vectors and for theirproduction wherein such vectors contain at least two surface componentsthat can confer tropism both for target cells and for packaging cells.The presence in these virus vectors of at least two such components canprovide independent capabilities for the efficient propagation of saidvector viruses in packaging cells and for the efficient gene delivery totarget cells. Affinity for the packaging cell provides for propagationto high yields by the ability of propagated vector viruses to re-infectpackaging cells and undergo repeated cycles of propagation.

A variety of compounds that can present themselves on the surface of avirus can be used for the purposes of this invention, and these can bederived from virus envelope or capsid proteins or from proteins derivedfrom other biological systems that have affinity for animal, plant orhuman cells and that can be incorporated into a virus vector surface.Such compounds useful for this purpose include protein molecules thatconsist of the natural amino sequence of such a protein or of a portionthereof. Such proteins or fragments can be modified in whole or in part.Proteins that can present themselves on the surface of a multitropicvirus vector can be chimeric molecules formed between a protein nativeto the vector virus or a fragment thereof and a protein non-native tosaid virus or a fragment thereof.

The proteins of multitropic vector can be native or non-native to saidvirus vector. Useful native proteins include the retrovirus ecotropicand amphotropic, polytropic or xenotropic env proteins. Non-nativeproteins useful for gene delivery to human cells include all of theenvelope proteins from human viruses, e.g., gp120 derived from HIV-1 orHIV-2 that can provide tropism for CD4+ cells, env proteins of HTLVI andHTLVII that can provide tropism for T cells, the envelop proteins ofhepatitis B virus (HBV) that can provide tropism for liver cells.Envelope proteins from influenza such as HA that can provide tropism tohuman cells can also be useful. Envelope protein from EBV can alsoprovide tropism for human B cells.

Multitropic vectors can be conveniently produced in packaging cells thatprovide for the synthesis of such components. These components willincorporate into the virus envelope in such a packaging cell. Nucleicacid sequences that provide for the synthesis of such compounds canreside on one or more plasmids and such plasmids can be introduced intopackaging cells. Such a nucleic acid construct can be present in anepisomal state or can be integrated into the genome of the packagingcell or can be in a combination of both episomal and integrated states.Packaging can be carried out by the methods and processes as disclosedherein.

Multitropic vectors can be prepared from such viruses as retroviruseswherein they contain in the virus envelope two or more compounds thatare native to said virus such as the ecotropic env protein of MMLV andthe amphotropic, polytropic or xenotropic env proteins of MMLV.Packaging of such a multitropic vector can be carried out bycompositions described above. The presence of the ecotropic env proteinin the virus envelope of the vectors can provide for efficientpropagation of said virus vector in packaging cells derived from mousecells, and the amphotropic, polytropic or xenotropic env protein canprovide for delivery to human cells.

For example, a packaging cell that can produce multitropic retrovirusvectors containing both the ecotropic env protein and an amphotropic,polytropic or xenotropic env protein can be made from a mouse packagingcell such as 3T3 cells. Such a packaging cell could be constructed bythe introduction into the cell of one or more plasmids containing thesequences encoding the packaging components, i.e., gag and pol, and thetwo envelope proteins. A cell line that highly expresses the packagingcomponents can be selected and cloned. The subsequent introduction ofprovirus vector DNA plasmid into such a packaging cell line can initiateproduction of multitropic vectors.

Multitropic virus vectors can also be prepared wherein such viruses areretroviruses that contain in the virus envelope two or more compounds,at least one of which is native to said virus, such as the ecotropic envprotein of MMLV, and at least one compound that is non-native to thevirus vector but has affinity to the target cell. Such a non-nativecompound as that can be derived from another virus envelope and furtherprovides affinity for the target cell. For example, a packaging cellthat can produce multitropic retrovirus vectors containing both a nativeecotropic env protein and a non-native protein such as the HIV-1 gp120can be produced in a mouse packaging cell such as a modified 3T3 cell.Such a multitropic virus vector can be produced as described above usingnucleic acid sequences for production of gp120 in place of nucleic acidsequences for the amphotropic, polytropic or xenotropic env protein. Thepresence of the ecotropic env protein in the virus envelope of thepropagated multitropic vectors can provide for efficient propagation ofsaid virus vector in packaging cells derived from mouse cells, and thegp120 protein can provide for delivery to CD4+ human cells. As describedjust above, the proteins of multitropic vector can be native ornon-native to said virus vector. Useful native proteins include theretrovirus ecotropic and amphotropic, polytropic or xenotropic envproteins. Non-native proteins useful for gene delivery to human cellsinclude all of the envelope proteins from human viruses, e.g., envproteins of HTLVI and HTLVII that can provide tropism for T cells, theenvelop proteins of hepatitis B virus (HBV) that can provide tropism forliver cells. Envelope proteins from influenza such as HA that canprovide tropism to human cells can also be useful. Envelope protein fromEBV can also provide tropism for human B cells. All of the foregoingcomponents could be propagated in a similar manner.

The present invention also provides compositions and methods of use forvirus vectors that contain in the virus envelope or in the virus capsidat least one component that is non-native to said virus but is native toa target cell. Such a component could be a ligand for a target cellreceptor. Efficient propagation of such a virus vector (hereindesignated as a monotropic virus vector) can be attained by the use of apackaging cell that is native to said virus and that has been modifiedto contain on its cell surface a receptor for the non-native viralcomponent.

Monotropic virus vectors thus possess the same advantages as multitropicvectors for the efficient delivery to a target cell. These vectorsexhibit the efficient adsorption similarly to a native virus to itstarget cell. These vectors lack, however, the undesirable risk to ahuman subject posed by a native virus. Thus, vector viruses such asanimal retroviruses like MMLV can provide the advantages of a nativevirus vector without its dangers. Thus, for example, monotropicproperties can provide for animal viruses to be utilized for genetransfer to human cells by the incorporation into the virus envelope oronto the virus surface of any of a wide variety of compounds non-nativeto said virus wherein such compounds provide tropism for certain typesof cells. Efficient propagation of monotropic viruses that lack a nativecompound for affinity to native packaging cells can be attained bymodifications made to such packaging cells. Thus, a cognate cellularreceptor or receptors corresponding to the non-native component which ispresent on the virus surface or associated with the virus envelope canbe introduced onto the surface of a packaging cell native to the virusvector. As an alternative approach, human cells for packaging of animalvirus can be developed by providing all sequences for packagingcomponents such as gag, pol and env from HIV (gp120).

A variety of compounds non-native to a virus vector that can presentthemselves on the surface of a virus or associate with the virusenvelope can be used for the preparation of monotropic virus vectors,and these can be derived from envelope proteins or from proteins thatprovide affinity for animal, plant or human cells that can be derivedfrom viruses or from other biological systems. Such compounds caninclude ligands derived from viruses, protein ligands derived from cellsand proteins or peptides that can provide fusion with cell membranes.Such compounds useful for this purpose include protein molecules whereinsuch proteins can consist of the entire amino sequence of such a proteinor of a portion thereof or wherein such proteins or fragments thereof orcontain partially modified sequences.

Propagation of monotropic virus vectors can be conveniently performed inpackaging cells that are native to the virus vector or native to aligand present in a virus vector wherein such cells have been modifiedby the introduction of one or more non-native components that can act asreceptors for monotropic viruses. Thus, an monotropic animal virusvector could, for example, contain a non-native ligand that provides fortropism to human cells. Propagation of said vector could be realized bypropagation in a packaging cell native to the virus vector that has beenmodified to contain on its surface a receptor corresponding to thenon-native ligand present on the monotropic virus. Such receptors can beintroduced into packaging cells by the incorporation of nucleic acidsequences that provide for the synthesis of such receptors. Such nucleicacid sequences could be present in the cell either in an integrated(into the cell genome) or in an episomal state. The propagation ofmonotropic virus vectors can proceed by the introduction into the cell(native or non-native cell) of nucleic acid sequences for the virusvector nucleic acid and for the packaging components, including thenon-native compound, wherein expression can proceed from these sequencespresent in an episomal state or in an integrated state

A monotropic virus vector, such as, for example, a retrovirus vectorsuch as MMLV, could be prepared to contain a non-native component (suchas gp120) that provides tropism for specific types of human cells aswell as for a modified mouse packaging cell. Such a vector will possesstropism for CD4+ human cells as well for modified packaging cell derivedfrom mouse 3T3 cells that have been modified by the incorporation ofnucleic acid sequences coding for CD4 and CCR-5 receptor proteins on thecell surface (Maddon, P. J., et al., Cell 47:33, 1986, incorporated byreference herein). Vector production could be carried out, for example,by a reverse packaging process as describe elsewhere in this patent bythe stable incorporation into the genome of said cell of nucleic acidsequences that code for vector nucleic acid sequence. Propagation can beinitiated by the introduction into such cells of nucleic acid sequencesthat provide synthesis of the packaging components of said retroviruswherein such nucleic acid sequences include sequences for gp120 but notfor a native env protein, i.e., neither the ecotropic or the amphotropicenv proteins native to MMLV. Such sequences can be present on plasmidsthat can be amplified such as described elsewhere in this patent toprovide for maximum synthesis of packaging components.

Packaging Systems

In general, the propagation of a viral vector (without a helper virus)proceeds in a packaging cell in which a nucleic acid sequence forpackaging components were stably integrated into the cellular genome andnucleic acid coding for viral nucleic acid is introduced in such a cellline. In such a system, the packaging components availability is alimiting element for packaging, which leads to low titter or loss ofcontinuous stability of nucleic acid sequence related to packagingcomponents, and could lead to a packaging cell incapable of viralproduction.

To overcome these limitations, the present invention provides methodsand compositions for novel reverse packaging systems that provide forefficient synthesis of packaging components without the use of helpervirus and may further reduce or eliminate the probability forrecombination events that can lead to the appearance of recombinationcompetent virus by use of cDNA of a gene fragment or by any methods tominimize overlapping sequences of plasmids carrying sequences coding forpackaging components.

Such a composition provided by this invention comprises a packaging cellwherein the nucleic acid sequence coding for the production of a virusvector nucleic acid components is stably integrated into the cell'sgenome. The packaging cell further provides all necessary packagingcomponents. The use of such a reverse packaging system can overcome thelimitations of other packaging systems by providing for optimalsynthesis of packaging components, which can be accomplished byamplified expression of packaging components following transfection orby compositions and the methods described in full detail below.

Optimal expression of packaging components in the packaging cell wherethe sequence coding for vector nucleic acid is stably integrated intothe cellular genome is achieved by introduction of a nucleic acidconstruct or constructs coding for packaging components. Such componentscould be native or non-native to the vector, or can be derived fromgenomic DNA or cDNA or any fragments thereof, or modification thereof.The nucleic acid construct coding for such packaging components could bepresent in packaging cell line in one or more copies.

Compositions for reverse packaging can provide optimal synthesis ofvirus vector packaging components by the use of nucleic acidamplification. Such amplification can be used in combination with highlyefficient promoters for expression of packaging components as describedabove. Useful elements for the amplification of nucleic sequences forvector virus packaging components include the origin of replication forSV40 virus (SV40 ori) and the origin of replication for Epstein-BarrVirus (EBV ori). These elements can act to amplify a plasmid or othernucleic acid entity which contains sequences for the expression ofvector components. Amplification of sequences by the use of plasmids orother nucleic acid entities whose replication is controlled by the SV40ori can be accomplished by the expression in a packaging cell oftrans-acting T-antigen, while amplification by the use of plasmids orother nucleic acid entities whose replication is controlled by EBV orican be accomplished by the expression of EBNA. Cells with properties forthe packaging of vector virus through amplification can be realizedusing EBV ori and EBNA.

For example, compositions for reverse packaging cells which utilizenucleic acid amplification can be prepared as described above forreverse packaging of virus vectors but wherein a packaging cell thatefficiently and stably produces virus vector nucleic acid is transfectedwith one or more nucleic acid constructs containing SV40 ori or EBV oriwherein such nucleic acid constructs provide for the synthesis ofpackaging components. The trans acting T antigen or EBNA proteins can beproduced from sequences present in the cell prior to transfection withsaid nucleic acid constructs or they can be present on said nucleic acidconstructs.

Compositions and methods of use for reverse packaging systems canprovide for greatly reducing or eliminating the possibility ofrecombination events among nucleic acid segments that encode virusvector nucleic acids and packaging components wherein such recombinationevents could give rise to replication competent viruses. This can beaccomplished by elimination of overlapping regions of virus genomebetween two such segments in packaging cells.

An example of a packaging system that markedly reduces or eliminatessuch possibility for recombination events and which can be used incombination with reverse packaging and/or amplification compositions asdescribed above for the propagation of retrovirus vectors can be made bycloning of the retrovirus sequences for gag, pol and env wherein thesequences for LTR are not included. The gag, pol and env sequences canbe prepared from cDNA preparations and cloned into a nucleic acidconstruct such as a plasmid. All such sequences can be cloned into thesame plasmid wherein they can be expressed from one or more promoters,or such sequences can be cloned into two or more plasmids wherein two ormore such plasmids are required to provide all of the required sequencesfor viral packaging components and wherein at least one promoter isrequired for expression in each plasmid.

A variety of non-native promoter/enhancer elements, along withpolyadenylation signal, can be used for driving cDNA expression.Promoters/enhancers which are highly efficient and either constitutiveor inducible can be used. These include but are not limited to promotersderived from cellular genes, such as the metallothionenpromoter/enhancer and the elongation factor (EF) promoter/enhancer, orpromoters derived from viruses such as CMV early promoters/enhancers,including the promoter/enhancer for the CMV E1 a gene, promoters derivedfrom bacteriophages such as T3, T7 and SP6 when expression of cognatepolymerases can be established in a packaging cell. The use ofpromoters/enhancers such as the metallothionen promoter/enhancer canprovide for the induction of expression of vector components inpackaging cells.

Cells suitable for packaging retroviruses can be transfected with aplasmid that contains sequences for the expression of vector nucleicacid and a stably transfected cell line producing vector nucleic acidcan be selected. Retrovirus vectors can be produced by transfection ofthis cell line with one or more nucleic acid constructs, such asplasmids, that provide for expression of packaging components. Thepropagation of retrovirus vectors can proceed from a transienttransfection or from a stable transfection with plasmids that providefor packaging components.

Non-Viral Specific Nucleic Acid Complexes (NVS Complexes)

The present invention provides compositions and methods of use fornon-viral specific nucleic acid complexes (NVS complexes) that can offersignificant advantages for the use of non-viral vectors in genedelivery. Such NVS complexes are composed of a nucleic acid componentand one or more specific binding proteins that bind to one or morespecific nucleic acid sequences in the nucleic acid construct. Previouscompositions for non-viral nucleic acid complexes for gene delivery haverelied on non-specific complexes between nucleic acid component andpolypeptides or polycationic polymers lipids. A wide variety of suchentities have been used wherein binding to the nucleic acid sequences isnon-specific and/or ionic. It is recognized, however, that suchnon-specific binding to nucleic acid can interfere with function of suchnucleic acid, such as transcription, integration, transport into thecell and/or into the nucleus and can have other interfering effectsincluding toxicity.

While non-viral nucleic acid complexes can provide significantadvantages for gene delivery, these advantages have not or cannot berealized by the use of non-specific nucleic acid complexes that rely onnon-sequence specific binding components. The present inventionovercomes these limitations by providing for specific complex formationbetween specific nucleic acid sequence and protein components whereinthe binding of protein molecules that provide useful properties for genetransfer can be localized to defined regions of the nucleic acidconstruct. Such localization of specific binding proteins in the nucleicacid construct can reduce or eliminate any interference with regions ofthe nucleic acid component that provide biological activity. The presentinvention also provides for the controlled displacement of such specificbinding proteins from their cognate binding sites wherein suchdisplacement can remove any possible interference with biologicalfunction or can release proteins that can provide useful function in thecell.

The present invention provides compositions and methods of use fornon-viral specific nucleic acid complexes (NVS Complexes) that, uponintroduction into a cell, are capable of biological function, i.e., geneexpression, transcription, translation, integration, intracellulartransport, production of a protein in a cell, production of a nucleicacid in a cell or interaction with a nucleic acid or protein in a cell.The present invention can provide significant advantages for non-viralvectors through the use of specific binding proteins that attach tocognate nucleic acid sequences in the vector nucleic acid component andcan render the construct capable of one or more of the followingproperties: 1) binding to a target cell, 2) providing for introductionof the nucleic acid component into cells, 3) providing for localizationto sites within a cell, 4) providing a signal for integration intocellular DNA, 5) providing enzymatic activity for replication and/orexpression of vector nucleic acid within the cell 6) providingprotection of the nucleic acid component from degradation both in vivoand in vitro. In the present invention one or more of the aboveproperties can be provided without substantially interfering withbiological function of said vector nucleic acid.

The present invention provides advantages over non-viral complexes thatrely on non-specific or ionic binding between nucleic acid andpolypeptides or lipids by the use of specific binding proteins that canrecognize specific nucleic acid sequences in the vector nucleic acidcomponent and thus provide the capability to segregate regions ofspecific protein binding from sequences in the nucleic acid componentthat provide biological function. Thus, one or more of the aboveproperties can be provided without substantially interfering withbiological function of the nucleic acid component. Such specificsequences are not an element or a part thereof of a gene expressioncassette such as a promoter sequence, but if promoter sequences are usedthen they are not involved in transcription but only function to bindpeptides. Transcription from such sequences can be limited or eliminatedby the use of inverted nucleic segments or inverted nucleotidesimmediately downstream from such promoter sequences.

The specific binding proteins of NVS complexes can further attachthrough fusion, conjugation, or complexing either directly or indirectlyto other moieties including natural or unnatural, modified or unmodifiedoligo- or polypeptides; polycations; natural or unnatural, modified orunmodified oligo- or polysaccharides; multimolecular complexes;inactivated viruses; lipids; and ligands. Such components can haveenzymatic activity such as polymerase activity or protein with anybiological function including transport and integration. The NVScomplexes of the present invention can provide for the delivery ofnucleic acid to eukaryotic cells including the cells of plants, humansand other mammals and to prokaryotic cells.

Specific binding protein molecules and their cognate nucleic acidsequences useful to the practice of this invention include: thebacteriophage λ repressor

TATCACCGC

ATAGTGGCG;

the bacteriophage 434 repressor

AC AAGAAAA

T GTTC T TT T;

the tryptophan repressor of E. coli

GTACTAGTT A

CATGATCAAT;

the Met J repressor of E. coli,

AGACGTCT

TCTGCAGA;

the lac repressor of E. coli,

T G G A AT T G T G A G C G G A T A A C A A T T

ACCTTAACACTCGC CTATT GTTAA; (SEQ ID NO:5)

the Engrailed gene regulator protein of Drosophila,

TAAT

ATTA;

the MATα2 yeast repressor protein,

CATGTAATT

GTACATTAA;

the CAP gene activator of E. coli

AAAAGTGTG ACAT

TTT T CACACTGTA; (SEQ ID NO:8)

the GAL4 yeast transcription activator,

CCGGAGGACAG

GGCCTCCT GTC;

the E2 papillomavirus transcription regulator,

ACCGACGTCGGT

TGGCTGCAGCCA;

the yeast GCN4 transcription regulator,

ATGATC

TACTAG;

the zif268 murine gene regulator,

GCGTGGGCG

CGCACCCGC;

the glucocorticoid receptor transcription modulator,

CAGAACATC

GTCTT GTAG;

the TFIID transcription initiation factor,

TATATAAA

ATATATTT.

Cognate sequences can be part of a nucleic acid construct and can bepresent at one or more sites in the nucleic acid construct wherein oneor more such sequences can be present at any one site. Thus the numberof such cognate nucleic acid sequences can be so arranged in order toachieve one or more objectives including nuclease resistance.Furthermore, the presence of multiple copies of such sequences in arepeated array can provide for a desired binding constant between thenucleic acid and a binding protein. Two or more such sequences can bepresent in a nucleic acid component to provide for association with twoor more different kinds of specific binding proteins.

Useful properties can be provided to NVS complexes by protein/nucleicacid interactions that can be dissociated in a controlled manner. Thus,for example, as a means of eliminating any interference of boundproteins with biological function of the nucleic acid component, adissociable specific binding protein can be bound to its cognatesequence in the nucleic acid component and, following contact of the NVScomplex with the target cell but prior to expression of biologicalfunction, said complex in the cell can be exposed to a molecule thatinduces dissociation. Such proteins as the lac repressor of E. coli andits cognate sequence are useful in this regard wherein dissociation canbe effected by an inducer such as an appropriate saccharide or IPTG. Itis preferred that when such a complex carrying another component thatneeds to bind to nucleic acid to provide further function, e.g., suchproteins as RNA polymerase or reverse transcriptase wherein such inducedrelease will further improve such function provided by the NVS vector.

Specific binding proteins can be modified by chemical modification or byattachment to a variety of ligands that can provide useful propertiesfor nucleic acid transfer to target cells. Such ligands or chemicalmodifications, being any chemical moiety, natural or synthetic, that canbe utilized in this invention include macromolecules greater than 20,000m.w. as well as small molecules less that 20,000 m.w. The ligand caninclude both macromolecules and small molecules. Macromolecules that canbe utilized include a variety of natural and synthetic polymersincluding peptides and proteins, nucleic acids, polysaccharides, lipids,synthetic polymers including polycations, polyanions and mixed polymers.Small molecules include oligopeptides, oligonucleotides,monosaccharides, oligosaccharides and synthetic polymers includingpolyanions, polycations, lipids and mixed polymers. Small molecules canalso include mononucleotides, oligonucleotides, oligopeptides,oligosaccharides, monosaccharides, lipids, sugars and other natural andsynthetic entities.

Ligands and chemical modifications can be utilized to provide fornucleic acid transfer to cells by providing such useful properties as 1)binding to a target cell, 2) providing for introduction of the nucleicacid component into cells, 3) providing for localization to sites withinthe cell 4) providing a signal for integration into cellular DNA, 5)providing enzymatic activity for replication and/or expression of vectornucleic acid within the cell by such proteins as DNA polymerase, RNApolymerase, reverse transcriptase, DNA ligase. 6) Proteins that protectthe nucleic acid component from degradation.

-   -   1) Cell targeting entities that can be utilized include:    -   a) antibodies to cellular surface components and epitopes    -   b) viruses, virus components of fragments of virus components        that have affinity for cellular surface components. These        include such proteins as the gp120 protein of HIV-1 or HIV-2        that binds to the CD4+ receptor of T4 lymphocytes (Lever 1995        British Medical Bulletin 51:149, the contents of which are        incorporated by reference).    -   c) ligands that have affinity for cell surfaces. These include        hormones, lectins, peptides and proteins, oligosaccharides and        polysaccharides. Two such ligands that could be used, for        example, are asialoorosomucoid that binds to the cellular        asialoglycoprotein receptor (Wu et a1989 J Biol Chem 269:16985,        the contents of which are incorporated by reference) and        transferrin that binds to transferrin cellular receptors (Wagner        et al. 1992 89:6099, also incorporated by reference),    -   d) polycations such as polylysine that bind non-specifically to        cell surfaces (Wu and Wu, U.S. Pat. No. 5,166,320 (contents        fully incorporated by reference) wherein the function of a        specific binding protein could be improved if the charge on the        nucleic acid is neutralized.    -   e) matrix proteins such as fibronectin that bind to hematopoetic        cells and other cells (Ruoslahti et al. 1981 J Biol Chem 2:7277,        the contents of which are incorporated by reference).    -   2) Entities that facilitate cellular uptake include inactivated        viruses such as adenovirus (Crisitiano et al. 1993 Proc Natl.        Acad. Sci. USA 90:2122) Curiel et al. 1991 Proc Natl Acad. Sci.        USA 88:8850): virus components such as the hemaglutinating        protein of influenza virus and a peptide fragment derived from        it, the hemagglutinin HA-2 N-terminal fusogenic peptide (Wagner        et al. 1992 Proc Natl. Acad. Sci. USA 89:7934). The contents of        each of the foregoing publications are incorporated by        reference.    -   3) Entities that confer cellular location include:    -   a) nuclear proteins such as histones    -   b) nucleic acid species such as the snRNAs U1 and U2 (which can        be conjugated to binding proteins in accordance with known        method, see, for example, Pergolizzi et al., U.S. patent        application Ser. No. 06/491,929, filed on May 5, 1983, now        abandoned the contents of which are incorporated herein by        reference) which associate with cytoplasmic proteins and        localize in the nucleus (Zieve and Sautereauj, 1990,        Biochemistry and Molecular Biology 25: 1, the contents of which        are incorporated by reference).    -   4) entities which facilitate incorporation into cellular nucleic        acid include:    -   a) proteins that function in integration of nucleic acid into        DNA. These include integrase site specific recombinases (Argos        et al. 1986 EMBO Journ 5:433, incorporated by reference) and    -   5) Entities such as nucleic acid polymerases that act to        replicate vector nucleic acid sequences. These include such        enzymes as reverse transcriptase, RNA polymerases such as        derived form E. coli, T7 bacteriophages, and other virus,        prokaryotic and eucaryotic systems.    -   6) Entities such as that provide protection of the nucleic acid        component from degradation both in vivo and in vitro. Chemical        modifications or ligands can be fused, attached or conjugated        directly or indirectly to specific binding proteins by covalent        or non-covalent methods to provide such properties as described        above. Thus a specific binding protein or a fragment thereof can        be fused to a protein, such as a ligand, or a fragment thereof,        wherein the fused protein is a chimeric molecule with properties        provided by both proteins. Such a fused molecule can be prepared        by the methods of recombinant DNA or by chemical synthesis. Such        fused proteins can also be prepared wherein three or more        proteins, or fragments thereof, can be fused to form a chimeric        protein molecule. Such proteins may contain useful properties        provided by each of the constituent protein entities, or one or        more such sequences can act as a connector between polypeptide        sequences that provide function. Covalent linkage of protein        ligands to specific binding proteins by direct linkages can be        by methods practiced in the art including direct or indirect        chemical attachment to reactive sites in a specific binding        protein. Such covalent attachment could also be indirect wherein        a specific binding protein can be attached to a protein that is,        in turn, modified by attachment to a compound or protein that        provides useful function. Non-covalent methods that can be        utilized include modification of the specific binding protein to        provide for direct or indirect and/or specific or non-specific        binding of useful molecules including antigen-antibody        interactions, receptor-ligand interaction, by hydrophobic        interaction, polyionic interaction. Thus a specific binding        protein could contain native properties for binding to an        antibody, or could be attached to contain a compound that can be        bound by an antibody. Such an antibody could, in turn, be        modified by attachment to a protein or other compound that        provides useful function. Specific binding proteins could also        be modified to contain ligands such as biotin that can provide        binding to proteins such as avidin or streptavidin. Other useful        ligands that can be used include lectins.

The nucleic acid component of an NVS complex can be DNA, RNA, acombination of RNA and DNA, e.g., a DNA-RNA hybrid or a chimeric nucleicacid such as a DNA-RNA chimera. The nucleic acid components of a NVScomplex can be single stranded, double stranded or triple stranded. Thenucleic acid component be circular, linear or branched and may take theform of any DNA or RNA, and it can contain both double stranded regionsand single regions. All or part of the nucleic acid component can becomposed of modified nucleic acid or nucleic acid analogues. All or partof the nucleic acid component can be prepared by chemical or enzymaticmethods.

Nucleic acid sequences recognized by specific binding proteins can bepresent in the nucleic acid component in one or more copies. More thanone kind of such a cognate sequence can be present in a nucleic acidcomponent in order to provide for binding of two or more different kindsof specific binding proteins. Multiple copies of cognate sequences canbe present in close proximity one to another such as in one or moretandem array or such sequences can be present at sites throughout thenucleic acid component.

Regions of biological activity in the nucleic acid component of NSVcomplexes can specify coding for RNA (such as antisense RNA orribozymes) or for RNA that can be translated into protein. Regions ofbiological activity in NVS complexes can contain sequences forhybridization with intracellular nucleic acid sequences, integrationinto cellular DNA, termination sequences, primer sites and promotersites.

A NVS complex can be prepared, for example, using a nucleic acidcomponent such as a plasmid that contains nucleic acid sequences thatcan provide biological function cell and cognate nucleic acid sequencesrecognized by a specific binding protein such as the lac inducer region(Lac i) that can provide for the binding of lac repressor protein.Sequences for the lac inducer region can be included in multiple copiesin order to provide for binding of multiple copies of lac repressorprotein. In order to avoid any interference of biological activity bythe lac repressor, the multiple copies of the lac inducer sequence canbe localized to a region of the plasmid that is separate from sequencesproviding biological function. The lac repressor protein for thesepurposes can be modified to provide to provide useful properties forgene transfer. Thus, the lac repressor could be modified to provide forbinding to a target cell by conjugating, fusing or complexing with aprotein that provides affinity for targeted cells. Thus, such proteinsas the gp120 protein derived from HIV-1 that has properties forattachment to CD4+ cells or the surface antigen of HBV that providesaffinity for liver cells could be used. Sattentau, Q. J. and Weiss, R.A., Cell 52:631-633 (1988); Robinson, W. S: Hepandnavividae and theirreplication in Field, B N (ed.), Virology, Vol. 2, Second ed., 1989;pages 2137-2169, incorporated herein by reference.

An NVS complex can also contain more than one kind of specific bindingprotein in order to provide additional functions. A NVS complex could beconstructed as described above wherein, in addition to the localizedmultiple copies of cognate sequences for lac repressor binding,additional regions of the nucleic acid component containing multiplecopies of other specific binding proteins such as, for example, thebacteriophage 434 repressor and the bacteriophage λ repressor. Thesesequences can be also included in the nucleic acid component whereinthey are present at sites separate from nucleic acid sequences providingbiological function. The 434 repressor can be modified by conjugation,fusion or complexing to a nuclear localizing entity such as U1 RNA andthe λ repressor can be modified by conjugation to an integrase in orderto assist integration into the cell genome. Oraigie, R. et al., Cell62:829-837 (19901, contents of which are incorporated herein byreference.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 Reconstitution of aCis Effect that Resulted from the Inactivation of the U3Promoter/Enhancer in a Heterologous Vector (Retrovirus Vector) by theUse of Irrelevant Sequence Replacement

A Heterologous Vector (retrovirus vector) was prepared in which the LTRpromoter and enhancer were inactivated wherein polyadenylation functionwas reconstituted. Replacements were made to a retroviral vectorsequence (FIG. 1) present in a plasmid (pENZ1). A 188 base pair DNAfragment in the 3′ LTR U3 enhancer was replaced by 188 base pairsderived from the bacterial Neo gene (neomycin phosphotransferase)sequence through PCR strategy. Two regions of the promoter, one of 2base pairs and one of 6 base pairs, were each replaced by restrictionenzyme recognition sequences of the same size througholigonucleotide-mediated site directed mutagenesis. The removed nativesequences and the non-native replacement sequences are shown in FIG. 2.

The replacements of the enhancer and promoter regions were confirmed byDNA sequencing of these regions following manufacture's instruction (USBand ABI).

A complete Neo sequence was incorporated into this Heterologous Vectorsequence. Heterologous Vector (retroviruses vector) were produced bytransfection of a packaging cell line (PA 317 and GP+E-86) with thevector DNA construct. The propagated Heterologous Vectors (retrovirusvectors) were assayed in a transducing titer measuring Neo transductantsof 3T3 cells. A titer of up to 10⁶ transducing particles per ml wasobtained.

Example 2 A Retroviral Vector with Inactivated Promoter/Enhancer whichContains a Non-Native Polyadenylation Signal (the Mouse Histone H2A614Gene)

The nucleic acid sequence of Heterologous Vector retrovirus present in aplasmid that contains a Neo gene in a region outside of the retrovirusvector nucleic acid sequence can be modified by (FIG. 3) by replacementof a 188 base pair region of the 3′ enhancer with 188 base pairs derivedfrom the bacterial Neo gene as described in Example 1. By the samemethods, the promoter sequence can be replaced with sequences for a stemloop processing signal derived from mouse histone H2A614 gene.Retrovirus vectors containing these modifications can be produced bytransfection of packaging cells with this plasmid vector and selectionof a producer cell line. Such retrovirus vectors can be used fordelivery of an Exogenous Nucleic Acid to a target cell wherein mRNAexpressed from Exogenous Nucleic Acid can be polyadenylated by using thedownstream element of both the non-native mouse histone H2kA614stem-loop processing signal and the retrovirus AATAA element.

Example 3 A Retroviral Vector with Inactivated Promoter/Enhancer whichContains a Non-Native Polyadenylation Signal (the Human G-CSF Gene withthe AATAAA and mRNA Destabilization Elements Removed)

A Heterologous Vector (retrovirus vector) can be constructed in whichthe 3′ LTR promoter and enhancer were inactivated wherein the endogenousretroviral polyadenylation site is used. Modifications to provideinactivation are made to a retroviral vector nucleic acid sequencepresent in a plasmid (pENZ-1). The region of the LTR containing thepromoter/enhancer and the endogenous retroviral polyadenylation signalupstream from the AATAAA element was replaced with a portion of anefficient exogenous polyadenylation signal. In this way, vector mRNA canbe polyadenylated by using the retroviral downstream AATAAA element.Here, a polyadenylation processing signal from the human G-CSF gene withthe AATAAA and mRNA destabilization elements removed can be used toreplace a region of the 3′ U3 that encompasses both the promoter andenhancer sequences (FIG. 4). In the case, the retroviral AATAAA elementis used.

Example 4 Transcription of Chimeric RNA from a Heterologous VectorRetrovirus Using a Promoter/Enhancer Recognized by Pol 1

A Heterologous Vector retrovirus can be constructed as described inExample 1 to deliver an Exogenous Nucleic Acid sequence that transcribesa chimeric molecule composed of an antisense RNA and rRNA (FIG. 5). Asequence such as Neo can be present residing outside of the HeterologousVector sequence in the plasmid to provide for selection of producercells. This vector construct is used to transfect packaging cells toproduce Heterologous Vector retroviruses. The Heterologous Vectorretroviruses are used to transduce a target cell. The polymerase I ofthe target cell provides for synthesis of the chimeric RNA fromintegrated Heterologous Vector DNA.

Example 5 Transcription of Chimeric RNA from a Heterologous VectorRetrovirus Using a Promoter/Enhancer Recognized by Pol III

A Heterologous Vector retrovirus can be constructed as described inExample 1 to deliver an Exogenous Nucleic Acid sequence that transcribesa chimeric molecule composed of an antisense RNA and tRNA (FIG. 6). Asequence such as Neo can be present in a region outside of theHeterologous Vector sequence in the plasmid to provide for selection ofproducer cells. This vector construct is used to transfect packagingcells to produce Heterologous Vector retroviruses. The HeterologousVector retroviruses are used to transduce a target cell. The polymeraseIII of the target cell provides for synthesis of the chimeric RNA fromintegrated Heterologous Vector DNA.

Example 6 Transcription of Chimeric RNA from a Heterologous VectorRetrovirus Using a Promoter/Enhancer Recognized by Pol 11 Wherein theTranscript is not Polyadenylated

A Heterologous Vector retrovirus can be constructed as described inExample 1 to deliver an Exogenous Nucleic Acid sequence that transcribesa chimeric molecule composed of an antisense RNA and the snRNA moleculeU1 (FIG. 7). A sequence such as Neo can reside in a region outside ofthe Heterologous Vector sequence in a plasmid to provide for selectionof producer cells. This vector construct is used to transfect packagingcells to produce Heterologous Vector retroviruses. The HeterologousVector retroviruses are used to transduce a target cell. The polymeraseII of the target cell provides for synthesis of the chimeric RNA fromintegrated Heterologous Vector DNA.

Example 7 Propagation of AAV Vector Viruses from a Heterologous VectorRetrovirus Containing Non-Native Vector Components Derived from AAV

A retroviral vector DNA construct can be constructed to contain two AAVITR sequences wherein one is inserted into a site immediately downstreamfrom the primer binding site and the other is inserted into a site justupstream from the retrovirus origin for second strand DNA synthesis(ppt) such that following reverse transcription in a cell the ITRsequences will be separated by approximately 4.5 kb (FIG. 8). AnExogenous Nucleic Acid can be inserted between the AAV ITR sequences.

The sequences for the AAV cap protein and for the retrovirus for reversetranscriptase function are inserted into a plasmid such as pBR322. Thesequences are expressed under the control of the promoter/enhancer of aconstitutive cellular gene such as the elongation factor (EF).

The retroviral vector DNA construct and the plasmid containing the capand the reverse transcriptase sequences are used to transfect apackaging cell. Transcripts of Heterologous Vector RNA are reversetranscribed to yield double stranded retrovirus vector DNA. The repproduct can mediate synthesis of single stranded vector DNA wherein therep sequences and the Exogenous Nucleic Acid are flanked by the AAV ITRsequences. The presence of cap protein provides for packaging of AAVvirus vectors.

Example 8 Propagation of AAV Vector Viruses from a Heterologous VectorRetrovirus Inactivated for ppt Function and Containing Non-Native VectorComponents Derived from AAV

A retroviral vector DNA construct can be constructed to contain two AAVITR sequences wherein one is inserted into a site immediately downstreamfrom the primer binding site and the other is inserted into a site fromwhich the ppt sequences were deleted. The AAV rep sequences can beinserted into a site between the ITR sequences (FIG. 9). An ExogenousNucleic Acid can also be inserted between the AAV ITR sequences.

The sequences for the AAV cap protein and for the retrovirus reversetranscriptase function are inserted into a plasmid such as pBR322. Thesequences are expressed under the control of the promoter/enhancer of aconstitutive cellular gene such as the elongation factor (EF).

The retroviral vector DNA construct and the plasmid containing the capand reverse transcriptase sequences are used to cotransfect orsequentially transfect cells. Transcripts of Heterologous Vector RNA arereverse transcribed to yield Heterologous Vector DNA which is singlestranded due to the lack of the ppt sequences. In the single strandedDNA product of reverse transcription the AAV ITRs are separated byapproximately 4.5 kb and they flank the rep sequence and the ExogenousNucleic Acid. The rep products can mediate synthesis of single strandedvector DNA wherein the rep sequences and the Exogenous Nucleic Acid areflanked by the AAV ITR sequences. The presence of cap protein providesfor packaging of AAV virus vectors.

Example 9 Propagation of AAV Vector Viruses from a Heterologous VectorRetrovirus wherein ppt is Deleted and AAV ITR Sequences Flank the PBSSite

A Heterologous Vector (retrovirus vector) DNA construct can be made tocontain two AAV ITR sequences that flank the primer binding site (PBS)(FIG. 10). The ppt sequences are removed and the AAV rep sequences areinserted in their place. A Exogenous Nucleic Acid can be insertedbetween the inserted rep sequence and the downstream ITR sequence.

The sequences for the AAV cap protein and for the retrovirus reversetranscriptase function are inserted into a plasmid such as pBR322. Thesegenes can be expressed under the control of the promoter/enhancer of aconstitutive cellular gene such as the elongation factor (EF).

The retroviral vector DNA construct and the plasmid containing thesequences for cap and reverse transcriptase function are used tocotransfect or sequentially transfect cells. Transcripts of HeterologousVector RNA are reverse transcribed to yield Heterologous Vector DNAwhich is single stranded due to the lack of the ppt sequences. The repproducts can mediate synthesis of single stranded vector DNA wherein therep sequences and the Exogenous Nucleic Acid are flanked by the AAV ITRsequences wherein the AAV ITR sequences are separated by approximately4.5 kb. The presence of cap protein provides for packaging of AAV virusvectors.

Example 10 A Heterologous Vector (Retrovirus Vector) that Provides forAAV-Directed Integration of Exogenous Nucleic Acid

A Heterologous Vector (retrovirus vector) can be constructed to containtwo AAV ITR sequences wherein one is inserted into a site immediatelydownstream from the primer binding site and the other is inserted into asite just upstream from the retroviral origin for second strand DNAsynthesis (ppt) (FIG. 11). The AAV rep sequences are inserted at a sitebetween the two AAV ITR sequences. A Exogenous Nucleic Acid can beinserted between the ITR sequences. Heterologous Vector (retrovirusesvector) are produced in retrovirus packaging cells such as the onesdescribed in this patent. The retrovirus vectors are used to transducetarget cells wherein the vector RNA undergoes reverse transcription toproduce a double stranded DNA. The AAV ITRs and the rep productexpressed from the Heterologous Vector can mediate synthesis of singlestranded vector DNA wherein the rep sequences and the Exogenous NucleicAcid are flanked by the AAV ITR sequences. The AAV rep also functions inintegration with site specificity for the q13.4-ter region of chromosome19 of a human target cell.

Example 11 A Heterologous Vector (Retrovirus Vector) with ppt Deletedthat Provides for AAV-Directed Integration of Exogenous Nucleic Acid

A Heterologous Vector (retrovirus vector) can be constructed to containtwo AAV ITR sequences wherein one is inserted into a site immediatelydownstream from the primer binding site and the other is used to replacethe sequences for retroviral origin for second strand DNA synthesis(ppt) (FIG. 12). The AAV rep sequences are inserted at a site betweenthe two AAV ITR sequences. A Exogenous Nucleic Acid can be insertedbetween the ITR sequences. Such Heterologous Vector (retrovirusesvector) are produced in retrovirus packaging cells such as the onesdescribed in this patent. The retrovirus vectors are used to transducetarget cells wherein the vector RNA undergoes reverse transcription toproduce single stranded Heterologous Vector DNA due to the lack of theppt sequences. The AAV ITRs and the rep product expressed from theHeterologous Vector can mediate synthesis of single stranded vector DNAwherein the rep sequences and the Exogenous Nucleic Acid are flanked bythe AAV ITR sequences. The AAV rep also functions in integration withsite specificity for the q13.4-ter region of chromosome 19 of a humantarget cell.

Example 12 A Heterologous Vector (Retrovirus Vector) that Provides forAAV-Directed Integration of Exogenous Nucleic Acid Wherein ppt isDeleted and AAV ITR Sequences Flank the PBS Site

A Heterologous Vector (retrovirus vector) can be constructed to containtwo AAV ITR sequences which flank the primer binding site (PBS) (FIG.13). The ppt sequences are removed and the AAV rep sequences areinserted in their place. A Exogenous Nucleic Acid can be insertedbetween the rep sequence and the downstream ITR sequence. SuchHeterologous Vector (retrovirus vectors) are produced in retroviruspackaging cells such as the ones described in this patent. Theretrovirus vectors are used to transduce target cells wherein the vectorRNA undergoes reverse transcription to produce single strandedHeterologous Vector DNA due to the lack of the ppt sequences. In thesingle stranded DNA product of reverse transcription the AAV ITRs flankthe rep sequence and the Exogenous Nucleic Acid. The AAV ITRs and therep product expressed from the Heterologous Vector can mediate synthesisof single stranded vector DNA. The AAV rep also functions in integrationwith site specificity for the q13.4-ter region of chromosome 19 of ahuman target cell.

Example 13 Use of a Heterologous Vector for the Delivery of ChimericAntisense RNA Directed Against HIV-1 to CD34 Cells in an Ex Vivo Format

A retrovirus vector can be prepared as described in Example 6 whichcontains a sequence for a chimeric RNA composed of U1 snRNA and anantisense sequence directed against HIV-1. The promoter and enhancerregions of the LTR can be inactivated as described in Example 1. Thevector can be constructed to contain no other Heterologous Nucleic Acidand thus cannot produce any protein.

Stromal cultures can be established from bone marrow collection frompatients. Cells are plated at a concentration of 3−5×10⁵ cells/ml inIMDM medium. After generation of the stromal layer, the stromal cellsare irradiated and plated at 5×10⁵ cells per T-25 vent-cap flask in IMDMcontaining 10% autologous serum on the day before use.

After successful establishment of the stromal culture, leukapheresis ofpatient blood can begin. Patients undergo leukapheresis followingpriming with hematapoetic growth factor GCSF by conventional methods.Before leukapheresis, 300 ml of blood is drawn and sterile serumprepared by conventional methods, plasma is collected and white bloodcells purified by standard ficoll separation methods. The leukapheresiscollections are further purified by Ficoll-Hypaque density gradientcentrifugation to separate the PBMC from red cells and neutrophils. TheBaxter Isolex procedure can be used to enrich the PBMC fraction forcells expressing CD34+ antigen (stem cells). These cells are eluted fromthe Baxter column into a tissue culture bag. Cellular phenotype(presence of CD34+ markers) is assessed by flow cytometry prior toexpansion.

The cells from the column are expanded in a cell-free, factor-freegrowth medium supplemented with 10% autologous serum, using theautologous stromal cells as a supporting layer. Stroma is not used untilthe fourth passage. At this point most hematapoetic cells can beeradicated except for mature macrophages (Nolta, J. A. et al. 1995,incorporated by reference herein). The autologous serum can be filtersterilized and determined to be free of mycoplasma, bacteria and fungi.

The cells are grown in the tissue culture bag for 72 hours. The cellsfrom some patients are grown in the absence of GCSF and the cells fromthe remaining patients are grown in the presence of GCSF. Beforetransduction, a sample of CD34+ enriched cells is removed forquantitative measurement of antisense DNA and RNA/cell using PCR andRT/PCR.

The transducing vector can be produced as FDA certified material byappropriate contractors. Vectors consist of high titer (104-106 colonyforming units per ml) supernatants of the packaging cell line, PA317.The supernatants are free of pathogens and helper virus.

Cells are resuspended at a concentration of 10⁵ per ml in a transductionmedium. The cells are transduced with the MMLV construct (describedabove) with inactivated 3′-terminal LTR and a sequence for production ofa chimeric U1/HIV-1 antisense. After adsorption, fresh medium is addedand the cells are grown for one week at 37° C. Aliquots can be stored atall stages.

A sample of CD34+ enriched cells is removed at this time forquantitative measurement of antisense DNA and RNA/cell using PCR andRT/PCR. The transduced cells are grown for 1 week in culture at 37° C.The number of the transduced cells are determined at the end of oneweek. Samples are prepared for phenotypic analysis. Samples for Gramstain and microbiologic cultures for aerobic and anaerobic bacteria andfungus will be obtained prior to infusion.

The transduced cells are harvested, washed 3 times in normal saline andresuspended in normal saline. The final cell preparation is filteredthrough a platelet filter and transferred into a transfusion pack forinfusion. Intravenous catheterization with standard sterile technique isperformed. The infusion can be of not more than 5×10⁸ cells/kg of bodyweight. Total volume of infused cells does not exceed 10 ml/kg of bodyweight. After an initial test infusion of 1-5% of the total volume,cells are infused over the next 60-120 minutes. During infusion, thecell suspension is mixed gently approximately every 5 minutes while thepatient is being observed for acute and subacute toxicity.

Patients are monitored for the production of CD4+ cells expressingU1/antisense RNA by RT-PCR as described (Liu, D. et al. 1997 J. Virol.in press, contents incorporated by reference) and for plasma virusconcentration and for CD4+ cell count.

Many obvious variations will no doubt be suggested to those of ordinaryskill in the art in light of the above detailed description and examplesof the present invention. All such variations are fully embraced by thescope and spirit of the invention as more particularly defined by theclaims that follow.

1. A packaging cell line for propagating a viral vector independent of ahelper virus, said viral vector comprising a nucleic acid component andat least two different non-nucleic components, wherein one of saidnon-nucleic acid components has a tropism for said cell line and theother non-nucleic acid component has a tropism for a target cell whichis different from said cell line, said nucleic acid component and saidnon-nucleic acid components being capable of forming a specific complexor complexes, wherein said sequence or sequences for the viral vectornucleic acid component have been stably integrated in the genome of saidcell line, and sequences for the non-nucleic acid components of saidviral vector have been stably integrated in the genome of said cell lineand code for envelope proteins from two different viruses.
 2. Thepackaging cell line of claim 1, wherein said viral vector comprises aretrovirus or retroviral sequences.
 3. The packaging cell line of claim1, wherein said viral vector nucleic acid component comprises nucleicacid sequences derived from genomic DNA, cDNA, or fragments of either orboth of the foregoing.
 4. The packaging cell line of claim 1, whereinsaid packaging cell line and said target cell are from differentspecies.
 5. The packaging cell line of claim 1, wherein said target cellcomprises T cells, liver cells, bone marrow cells, epithelial cells, ora combination of any of the foregoing.
 6. The packaging cell line ofclaim 1, wherein the viral vector produced from said packaging cell linecodes for a protein of interest that is expressed in said target cell.7. The packaging cell line of claim 1 wherein the viral vector producedfrom said packaging cell line codes for an antisense RNA that istranscribed in said target cell.
 8. The packaging cell line of claim 1,wherein the viral vector produced from said packaging cell line codesfor a protein of interest that is expressed in said target cell and foran antisense RNA that is transcribed in said target cell.
 9. Thepackaging cell line of claim 1, wherein said nucleic acid componentcomprises sequences derived from a virus that has a tropism to said cellline.
 10. The packaging cell line of claim 1, wherein said nucleic acidcomponent comprises sequences derived from a virus that has a tropism tosaid target cell.
 11. The packaging cell line of claim 1, wherein saidnucleic acid component comprises sequences derived from a virus that hasa tropism to said cell line and sequences derived from a different virusthat has a tropism to said target cell.
 12. The packaging cell line ofclaim 4, wherein said packaging cell line is a non-human animal speciesand said target cell is human.
 13. The packaging cell line of claim 12,wherein said non-human animal species is murine.
 14. The packaging cellline of claim 7, wherein said antisense RNA is complementary to an mRNAcoding for a undesirable protein in said target cell.
 15. The packagingcell line of claim 7, wherein said antisense RNA is part of a chimericRNA molecule that comprises sequences from small nuclear RNAs (snRNAs).16. The packaging cell line of claim 7, wherein said antisense RNA iseither (i) complementary to an mRNA coding for a undesirable protein insaid target cell or (ii) is part of a chimeric RNA molecule thatcomprises sequences from small nuclear RNAs (snRNAs).
 17. The packagingcell line of claim 8, wherein said antisense RNA is complementary to anmRNA coding for a undesirable protein in said target cell.
 18. Thepackaging cell line of claim 8, wherein said antisense RNA is part of achimeric RNA molecule that comprises sequences from small nuclear RNAs(snRNAs).
 19. The packaging cell line of claim 15, wherein said snRNAscomprises U1, U2, U3, U4, U5, U6, U7, U8, U9, U10 or U11.
 20. Thepackaging cell line of claim 18, wherein said snRNAs comprises U1, U2,U3, U4, U5, U6, U7, U8, U9, U10 or U11.
 21. The packaging cell line ofclaim 16, wherein said snRNAs comprises U1, U2, U3, U4, U5, U6, U7, U8,U9, U10 or U11.
 22. A packaging cell line for propagating a viral vectorindependent of a helper virus, said viral vector comprising a nucleicacid component and at least two different non-nucleic components,wherein one of said non-nucleic acid components has a tropism for saidcell line and the other non-nucleic acid component has a tropism for atarget cell which is different from said cell line, said nucleic acidcomponent and said non-nucleic acid components being capable of forminga specific complex or complexes, and wherein sequences for thenon-nucleic acid components of said viral vector have been stablyintegrated in the genome of said cell line and code for envelopeproteins from two different viruses.
 23. The packaging cell line ofclaim 22, wherein said viral vector comprises a retrovirus or retroviralsequences.
 24. The packaging cell line of claim 22, wherein saidpackaging cell line and said target cell are from different species. 25.The packaging cell line of claim 22, wherein said packaging cell line isa non-human animal species and said target cell is human.
 26. Thepackaging cell line of claim 22, wherein said target cell comprises Tcells, liver cells, bone marrow cells, epithelial cells, or acombination of any of the foregoing.
 27. The packaging cell line ofclaim 25, wherein said non-human animal species is murine.